The leaf microbiome plays an important role in plant health and defence. Despite its importance, how the assembly of the leaf microbial community is modified by environmental conditions such as nutrient availability remains relatively uninvestigated. Soil nutrient availability may shift the outcome of microbial interactions within a host individual or influence the pool of microbes across the plant community. We hypothesised that leaf microbial diversity would increase across the season as leaves collect additional taxa, and that this seasonal assembly would be sensitive to nutrient addition. To assess this, we tracked the assembly of the fungal phyllosphere microbiome of the grass tall fescue (Lolium arundinaceum) in old-field vegetation over the growing season and experimentally tested whether the seasonality of the microbiome was modified by experimental addition of soil nutrients. Fungal diversity (Shannon diversity index, richness, and evenness) increased early in the season, with most metrics saturating before the end of the season. Community composition as measured by Bray-Curtis dissimilarity also shifted over the early and mid-growing season. Phylogeny-based machine-learning identified fungal lineages that were abundant in different seasons, linking seasonal community shifts to their evolutionary context. Nutrient addition was less important than time of season, but still significantly altered community composition and interacted with time to influence richness, with lowest richness in the low nutrient addition plots early in the season. The clear seasonality of the microbiome provides support for a dynamic phyllosphere microbiome, suggesting further studies manipulating fungal recruitment over the season. Furthermore, it highlights the robustness of seasonal assembly to variation in nutrient availability.

ABSTRACT The outcomes of pathogen infection can be sensitive to temperature, interactions with other pathogen species, and host age. Yet few studies have experimentally tested how warming alters infection outcomes for multiple, potentially interacting, pathogen species across host ages. In this study, we conducted a factorial experiment to test how plant age (7‐week vs. 13‐week plants) and temperature (21°C vs. 29°C) influence infection outcomes of two foliar fungal pathogens with contrasting feeding strategies—Rhizoctonia solani (a necrotroph) and Colletotrichum cereale (a hemibiotroph)—in the grass species tall fescue (Lolium arundinaceum). Contrary to expectations of coinfection with pathogens of opposing life‐history strategies leading to increased disease symptoms, coinfection had relatively minor effects across disease metrics. Instead, infection outcomes were driven by host age, pathogen identity and temperature. In plants inoculated with R. solani, higher temperature reduced lesion severity, and independently, severity was less in older plants. C. cereale lesion development showed a strong age × temperature interaction, with older plants being more resistant to disease in cooler conditions but not under warming. In plants co‐inoculated with both pathogens, elevated temperature reduced disease severity, and this effect was stronger in older plants. These findings demonstrate that environmental conditions and host age can both interact and outweigh within‐host pathogen interactions, highlighting the importance of incorporating host demographic structure in predicting disease responses to climate warming.

Many disease epidemics recur seasonally, and such seasonal epidemics can be shaped by species interactions among parasites, pathogens, or other microbes. Field experiments are a classic approach for understanding species interactions but are rarely used to study seasonal epidemics. Our research objective was to help fill this gap by manipulating the seasonal timing of the establishment of infectious diseases while tracking epidemics and other ecological responses. To do this, we conducted a multiyear field experiment in an old field in the Piedmont of North Carolina, USA, dominated by the grass species tall fescue (Lolium arundinaceum (Schreb). Darbysh). In the field, tall fescue experienced seasonal epidemics of multiple foliar fungal diseases: anthracnose in spring, brown patch in mid-summer, and crown rust in late summer to fall. In a fully randomized design, we applied four fungicide treatments to replicate plots of intact vegetation in specific seasons to manipulate the timing of disease epidemics. One treatment was designed to delay the establishment of anthracnose until mid-summer, and another to delay the establishment of both anthracnose and brown patch until fall. In a third treatment, fungicide was applied year-round, and, in a fourth treatment, fungicide was never applied. The experiment comprised 64 plots, each 2 m × 2 m, surveyed from May 2017 to February 2020. Here, we report a dataset documenting responses in the community structure of both plants and foliar fungi. To track disease prevalence in the host population across seasons and years, this dataset includes monthly leaf-level observations of the disease status of over 100,000 leaves. To quantify transmission and investigate within-host pathogen interactions, we longitudinally surveyed disease status in host individuals of known age at least weekly over two growing seasons. Finally, the dataset includes annual data on infection prevalence of the systemic fungal endophyte Epichloë coenophiala, community-level aboveground plant biomass, and plant community cover. These data can be used for meta-analyses, comparisons, and syntheses across systems as ecologists seek to predict and mechanistically understand seasonal disease epidemics. There are no copyrights on the dataset, and we request that users of this dataset cite this paper in all publications resulting from its use.

Nutrient supply can amplify disease epidemics through mechanisms from individual to community levels. Within host individuals, nutrient addition can drive pathogen replication or growth. Across a host population, nutrient addition can drive disease transmission by increasing host growth and abundance relative to defense. Furthermore, such effects may be influenced by pathogen species interactions. Understanding how nutrients impact disease epidemics requires a framework that integrates these mechanisms across biological levels. To build such a framework, we conducted a field experiment in an old field on tall fescue, Lolium arundinaceum, and used structural equation models to integrate multiple hypothesized mechanisms. Nutrient addition (NPK fertilizer) increased brown patch disease but was best modeled as a direct path and not mediated by host abundance. To expand our framework, we also re-analyzed a previous experiment. That experiment reproduced the direct path from nutrients to disease, and added an indirect path mediated by host population abundance. Nutrient addition also increased foliar nitrogen, consistent with individual-level mechanism, but this did not increase disease. Brown patch decreased with burden of another disease, anthracnose, independently of nutrients. These results partially support both individual- and population-level hypotheses, emphasizing the importance of considering multiple biological levels underlying impacts of abiotic change. ### Competing Interest Statement The authors have declared no competing interest.

A pathogen arriving on a host typically encounters a diverse community of microbes that can shape priority effects, other within-host interactions and infection outcomes. In plants, environmental nutrients can drive trade-offs between host growth and defence and can mediate interactions between co-infecting pathogens. Nutrients may thus alter the outcome of pathogen priority effects for the host, but this possibility has received little experimental investigation. To disentangle the relationship between nutrient availability and co-infection dynamics, we factorially manipulated the nutrient availability and order of arrival of two foliar fungal pathogens ( Rhizoctonia solani and Colletotrichum cereale ) on the grass tall fescue ( Lolium arundinaceum ) and tracked disease outcomes. Nutrient addition did not influence infection rates, infection severity or plant biomass. Colletotrichum cereale facilitated R. solani , increasing its infection rate regardless of their order of inoculation. Additionally, simultaneous and C. cereale -first inoculations decreased plant growth and—in plants that did not receive nutrient addition—increased leaf nitrogen concentrations compared to uninoculated plants. These effects were partially, but not completely, explained by the duration and severity of pathogen infections. This study highlights the importance of understanding the intricate associations between the order of pathogen arrival, host nutrient availability and host defence to better predict infection outcomes.

The structure of the leaf microbiome can alter host fitness and change in response to abiotic and biotic factors, like seasonality, climate, and leaf age. However, relatively few studies consider the influence of host age on microbial communities at a time scale of a few days, a short time scale relevant to microbes. To understand how host age modulates changes in the fungal and bacterial leaf microbiome on a short time scale, we ran independent field and greenhouse-based studies and characterized phyllosphere communities using next-generation sequencing approaches. Our field study characterized changes in the fungal and bacterial phyllosphere by examining leaves of different relative ages across individuals, whereas the greenhouse study examined changes in the fungal microbiome by absolute leaf age across individuals. Together, these results indicate that fungal communities are susceptible to change as a leaf ages as evidenced by shifts in the diversity of fungal taxa both in the field and the greenhouse. Similarly, there were increases in the diversity of fungal taxa by leaf age in the greenhouse. In bacterial communities in the field, we observed changes in the diversity, composition, and relative abundance of common taxa. These findings build upon previous literature characterizing host-associated communities at longer time scales and provide a foundation for targeted work examining how specific microbial taxa might interact with each other, such as fine-scale interactions between pathogenic and non-pathogenic species.

Host individuals are commonly coinfected with multiple parasite species that may interact to shape within-host parasite community structure. In addition to within-host species interactions, parasite communities may also be structured by other processes like dispersal and ecological drift. The timing of dispersal (in particular, the temporal sequence in which parasite species infect a host individual) can alter within-host species interactions, setting the stage for historical contingency by priority effects, but how persistently such effects drive the trajectory of parasite community assembly is unclear, particularly under continued dispersal and ecological drift. We tested the role of species interactions under continued dispersal and ecological drift by simultaneously inoculating individual plants of tall fescue with a factorial combination of three symbionts (two foliar fungal parasites and a mutualistic endophyte), then deploying the plants in the field and tracking parasite communities as they assembled within host individuals. In the field, hosts were exposed to continued dispersal from a common pool of parasites, which should promote convergence in the structure of within-host parasite communities. Yet, analysis of parasite community trajectories found no signal of convergence. Instead, parasite community trajectories generally diverged from each other, and the magnitude of divergence depended on the initial composition of symbionts within each host, indicating historical contingency. Early in assembly, parasite communities also showed evidence of drift, revealing another source of among-host divergence in parasite community structure. Overall, these results show that both historical contingency and ecological drift contributed to divergence in parasite community assembly within hosts.

Disease may drive variation in host community structure by modifying the interplay of deterministic and stochastic processes that shape communities. For instance, deterministic processes like ecological selection can benefit species less impacted by disease. When communities have higher levels of disease and disease consistently selects for certain host species, this can reduce variation in host community composition. On the other hand, when host communities are less impacted by disease and selection is weaker, stochastic processes (e.g., drift, dispersal) may play a bigger role in host community structure, which can increase variation among communities. While effects of disease on host community structure have been quantified in field experiments, few have addressed the role of disease in modulating variation in structure among host communities. To address this, we conducted a field experiment spanning three years, using a tractable system: foliar fungal pathogens in an old-field grassland community dominated by the grass Lolium arundinaceum, tall fescue. We reduced foliar fungal disease burden in replicate host communities (experimental plots in intact vegetation) in three fungicide regimens that varied in the seasonal duration of fungicide treatment and included a fungicide-free control. We measured host diversity, biomass, and variation in community structure among replicate communities. Disease reduction generally decreased plant richness and increased aboveground biomass relative to communities experiencing ambient levels of disease. These changes in richness and aboveground biomass were consistent across years despite changes in structure of the plant communities over the experiment's three years. Importantly, disease reduction amplified host community variation, suggesting that disease diminished the degree to which host communities were structured by stochastic processes. These results of experimental disease reduction both highlight the potential importance of stochastic processes in plant communities and reveal the potential for disease to regulate variation in host community structure.

Plant damage by invertebrate herbivores and pathogens influences the dynamics of grassland ecosystems, but anthropogenic changes in nitrogen and phosphorus availability can modify these relationships. Using a globally distributed experiment, we describe leaf damage on 153 plant taxa from 27 grasslands worldwide, under ambient conditions and with experimentally elevated nitrogen and phosphorus. Invertebrate damage significantly increased with nitrogen addition, especially in grasses and non‐leguminous forbs. Pathogen damage increased with nitrogen in grasses and legumes but not forbs. Effects of phosphorus were generally weaker. Damage was higher in grasslands with more precipitation, but climatic conditions did not change effects of nutrients on leaf damage. On average, invertebrate damage was relatively higher on legumes and pathogen damage was relatively higher on grasses. Community‐weighted mean damage reflected these functional group patterns, with no effects of N on community‐weighted pathogen damage (due to opposing responses of grasses and forbs) but stronger effects of N on community‐weighted invertebrate damage (due to consistent responses of grasses and forbs). Synthesis . As human‐induced inputs of nitrogen and phosphorus continue to increase, understanding their impacts on invertebrate and pathogen damage becomes increasingly important. Our results demonstrate that eutrophication frequently increases plant damage and that damage increases with precipitation across a wide array of grasslands. Invertebrate and pathogen damage in grasslands is likely to increase in the future, with potential consequences for plant, invertebrate and pathogen communities, as well as the transfer of energy and nutrients across trophic levels.

The interactions among host-associated microbes and parasites can have clear consequences for disease susceptibility and progression within host individuals. Yet, empirical evidence for how these interactions impact parasite transmission between host individuals remains scarce. We address this scarcity by using a field mesocosm experiment to investigate the interaction between a systemic fungal endophyte, Epichloë coenophiala, and a fungal parasite, Rhizoctonia solani, in leaves of a grass host, tall fescue (Lolium arundinaceum). Specifically, we investigated how this interaction impacted transmission of the parasite under field conditions in replicated experimental host populations. Epichloë-inoculated populations tended to have greater disease prevalence over time, though this difference had weak statistical support. More clearly, Epichloë-inoculated populations experienced higher peak parasite prevalences than Epichloë-free populations. Epichloë conferred a benefit in growth; Epichloë-inoculated populations had greater aboveground biomass than Epichloë-free populations. Using biomass as a proxy, host density was correlated with peak parasite prevalence, but Epichloë still increased peak parasite prevalence after controlling for the effect of biomass. Together, these results suggest that within-host microbial interactions can impact disease at the population level. Further, while Epichloë is clearly a mutualist of tall fescue, it may not be a defensive mutualist in relation to Rhizoctonia solani.

The initial colonization of a host by symbionts, ranging from parasites to mutualists, can generate priority effects that alter within-host interactions and the trajectory of parasite community assembly. At the same time, variation in parasite communities among hosts can also stem from stochastic processes. Community ecology theory posits that multiple processes (e.g. dispersal, selection and drift) interact to generate variation in community structure, but these processes are rarely considered simultaneously during community assembly. To test the role of these processes in a parasite community, we experimentally simulated dispersal of three symbionts by factorially inoculating individual plants of tall fescue with two foliar fungal parasites, Colletotrichum cereale and Rhizoctonia solani , and a hypothesized mutualist endophyte, Epichloë coenophiala . We then tracked parasite infections longitudinally in the field. After the initial inoculations, hosts were exposed to a common pool of parasites in the field, which we expected to cause parasite communities to converge towards a similar community state. To test for convergence, we analyzed individual hosts’ parasite community trajectories in multivariate space. In contrast to our expectation, there was no signal of convergence. Instead, parasite community trajectories generally diverged over time between treatment groups and the magnitude of divergence depended on the symbiont species inoculated. Parasite communities of hosts that were inoculated with only the mutualist, Epichloë , showed significant trends of divergence relative to all other symbiont inoculation treatments. In contrast, hosts inoculated with only Rhizoctonia did not exhibit clear trends of divergence when compared to other parasite inoculations. Further, co-inoculation with both parasite species resulted in faster rates of divergence and greater temporal change in parasite communities relative to hosts inoculated with only the parasite Colletotrichum . As predicted by existing theory, parasite communities showed evidence of drift during the beginning of the experiment, which contributed to among-host divergence in parasite community structure. Overall, these data provide evidence that initial dispersal of symbionts produced persistent changes in parasite community structure via ecological selection, that drift was important during the early stages of parasite community assembly, and together, dispersal, selection and drift resulted in parasite community divergence. Open Research statement The data and code that support the findings of this study are available through Zenodo at https://doi.org/10.5281/zenodo.5714452

Parasites can affect and be affected by the host's microbiome, with consequences for host susceptibility, parasite transmission, and host and parasite fitness. Yet, two aspects of the relationship between parasite infection and host microbiota remain little understood: the nature of the relationship under field conditions, and how the relationship varies among parasites. To overcome these limitations, we performed a field survey of the within-leaf fungal community in a tall fescue population. We investigated how diversity and composition of the fungal microbiome associate with natural infection by fungal parasites with different feeding strategies. A parasite's feeding strategy affects both parasite requirements of the host environment and parasite impacts on the host environment. We hypothesized that parasites that more strongly modify niches available within a host will be associated with greater changes in microbiome diversity and composition. Parasites with a feeding strategy that creates necrotic tissue to extract resources (necrotrophs) may not only have different niche requirements, but also act as particularly strong niche modifiers. Barcoded amplicon sequencing of the fungal ITS region revealed that leaf segments symptomatic of necrotrophs had lower fungal diversity and distinct composition compared to segments that were asymptomatic or symptomatic of other parasites. There were no clear differences in fungal diversity or composition between leaf segments that were asymptomatic and segments symptomatic of other parasite feeding strategies. Our results motivate future experimental work to test how the relationship between the microbiome and parasite infection is impacted by parasite feeding strategy and highlight the potential importance of parasite traits.

Interactions among parasites and other microbes within hosts can impact disease progression, yet study of such interactions has been mostly limited to pairwise combinations of microbes. Given the diversity of microbes within hosts, indirect interactions among more than two microbial species may also impact disease. To test this hypothesis, we performed inoculation experiments that investigated interactions among two fungal parasites, Rhizoctonia solani and Colletotrichum cereale, and a systemic fungal endophyte, Epichloë coenophiala, within the grass, tall fescue ( Lolium arundinaceum ). Both direct and indirect interactions impacted disease progression. While the endophyte did not directly influence R. solani disease progression or C. cereale symptom development, the endophyte modified the interaction between the two parasites . The magnitude of the facilitative effect of C. cereale on the growth of R. solani tended to be greater when the endophyte was present. Moreover, this interaction modification strongly affected leaf mortality. For plants lacking the endophyte, parasite co-inoculation did not increase leaf mortality compared to single-parasite inoculations. By contrast, for endophyte-infected plants, parasite co-inoculation increased leaf mortality compared to inoculation with R. solani or C. cereale alone by 1.9 or 4.9 times, respectively. Together, these results show that disease progression can be strongly impacted by indirect interactions among microbial symbionts.

Interactions among parasites and other microbes within hosts can impact disease progression, yet study of such interactions has been mostly limited to pairwise combinations of microbes. Given the diversity of microbes within hosts, higher-order interactions among more than two microbial species may also impact disease. To test this hypothesis, we performed inoculation experiments that investigated interactions among two fungal parasites, Rhizoctonia solani and Colletotrichum cereale, and a systemic fungal endophyte, Epichloë coenophiala, within a grass host. Both pairwise and higher-order interactions impacted disease progression. While the endophyte did not directly influence R. solani growth or C. cereale symptom development, the endophyte modified the interaction between the two parasites. The magnitude of the facilitative effect of C. cereale on the growth of R. solani tended to be greater when the endophyte was present. Moreover, this interaction modification strongly affected leaf mortality. For plants lacking the endophyte, parasite co-inoculation did not increase leaf mortality compared to single-parasite inoculations. In contrast, for endophyte-infected plants, parasite co-inoculation increased leaf mortality compared to inoculation with R. solani or C. cereale alone by 1.9 or 4.9 times, respectively. Together, these results show that disease progression can be strongly impacted by higher-order interactions among microbial symbionts.

Parasites can affect and be affected by the host’s microbiome, with consequences for host susceptibility, parasite transmission, and host and parasite fitness. Yet, there are two aspects of the relationship between parasite infection and the host microbiome that remain little understood: the nature of the relationship under field conditions, and how the relationship varies among parasite species. To overcome these limitations, we assayed the within-leaf fungal community in a grass population to investigate how diversity and composition of the fungal microbiome are associated with natural infection by fungal parasites with different feeding strategies. We hypothesized that parasites that more strongly modify niches available within a host will thereby alter the microbial taxa that can colonize the community and be associated with greater changes in microbiome diversity and composition. A parasite that creates necrotic tissue to extract resources (necrotrophs) may act as a particularly strong niche modifier whereas one that does not (biotrophs) may not. Barcoded amplicon sequencing of the fungal ITS region revealed that the microbiome of leaf segments that were symptomatic of necrotrophs had lower fungal diversity and distinct composition compared to segments that were asymptomatic or symptomatic of other parasites. There were no clear differences in fungal diversity or composition between leaf segments that were asymptomatic and segments that were symptomatic of other parasite feeding strategies. This supports the hypothesis that within-host niches link infection by parasites to the host’s microbiome. Together, these results highlight the importance of parasite traits in determining parasite impacts on the host’s microbiome.

Two key knowledge gaps currently limit the development of more predictive and general models of pathogen transmission: (1) the physiological basis of heterogeneity in host contribution to pathogen transmission (reservoir potential) remains poorly understood and (2) a general means of integrating the ecological dynamics of host communities has yet to emerge. If the traits responsible for differences in reservoir potential also modulate host community dynamics, these traits could be used to predict pathogen transmission as host communities change. In two greenhouse experiments, across 23 host species and two levels of resource supply, the reservoir potential of plant hosts increased significantly along the Leaf Economics Spectrum, a global axis of plant physiological trait covariation that features prominently in models of plant community ecology. This indicates that the traits of the Leaf Economics Spectrum underlie broad differences in reservoir potential across host species and resource supplies. Therefore, host traits could be used to integrate epidemiological models of pathogen transmission with ecological models of host community change.

Host and parasite richness are generally positively correlated, but the stability of this relationship in response to global change remains poorly understood. Rapidly changing biotic and abiotic conditions can alter host community assembly, which in turn, can alter parasite transmission. Consequently, if the relationship between host and parasite richness is sensitive to parasite transmission, then changes in host composition under various global change scenarios could strengthen or weaken the relationship between host and parasite richness. To test the hypothesis that host community assembly can alter the relationship between host and parasite richness in response to global change, we experimentally crossed host diversity (biodiversity loss) and resource supply to hosts (eutrophication), then allowed communities to assemble. As previously shown, initial host diversity and resource supply determined the trajectory of host community assembly, altering post-assembly host species richness, richness-independent host phylogenetic diversity, and colonization by exotic host species. Overall, host richness predicted parasite richness, and as predicted, this effect was moderated by exotic abundance-communities dominated by exotic species exhibited a stronger positive relationship between post-assembly host and parasite richness. Ultimately, these results suggest that, by modulating parasite transmission, community assembly can modify the relationship between host and parasite richness. These results thus provide a novel mechanism to explain how global environmental change can generate contingencies in a fundamental ecological relationship-the positive relationship between host and parasite richness.

High-resource environments typically favor quick-growing, poorly defended plants, while resource-poor environments typically favor slow-growing, well-defended plants. The prevailing hypothesis explaining this pattern states that, as resource availability increases, well-defended, slow-growing species are replaced by poorly defended, fast-growing species. A second hypothesis states that greater resource availability increases allocation to growth at the expense of defense, within species. Regardless of mechanism, if exotic species are released from enemies relative to natives, shifts in allocation to growth and defense both within and among species could differ by geographic provenance. To test whether resource availability alters growth or defense, within and among species, and whether any such effects differ between natives and exotics, we manipulated soil nutrient supply and access of aboveground insect herbivores and fungal pathogens under field conditions to individuals of six native and six exotic grass species that co-occurred in a North Carolina old field. The prevailing hypothesis’ prediction—that species-level enemy impact increases with species’ nutrient responsiveness—was confirmed. Moreover, this relationship did not differ between native and exotic species. The second hypothesis’ prediction—that individual-level enemy impact increases with nutrient supply, after accounting for species-level variation in performance—was not supported. Together, these results support the idea, across native and exotic species, that plant species turnover is the primary mechanism underlying effects of nutrient enrichment on allocation to growth and defense in plant communities.

Host and parasite richness are generally positively correlated, but the stability of this relationship during community assembly remains untested. The composition of host communities can alter parasite transmission, and the relationship between host and parasite richness is sensitive to parasite transmission. Thus, changes in composition during host community assembly could strengthen or weaken the relationship between host and parasite richness. Host community assembly, in turn, can be driven by many processes, including resource enrichment. To test the hypothesis that host community assembly can alter the relationship between host and parasite richness, we experimentally crossed host diversity and resource supply to hosts, then allowed communities to assemble. As previously shown, initial host diversity and resource supply determined the trajectory of host community assembly, altering post-assembly host species richness, richness-independent host phylogenetic diversity, and colonization by exotic host species. Throughout community assembly, host richness predicted parasite richness. As predicted, this effect was moderated by exotic abundance: communities dominated by exotic species exhibited a stronger positive relationship between post-assembly host and parasite richness. Ultimately, these results suggest that, by modulating parasite transmission, community assembly can modify the relationship between host and parasite richness, providing a novel mechanism to explain contingencies in this relationship.