Foliar Fungal Communities Research Articles

Leaf resistance traits influence endophytic fungi colonization and community composition in a South American temperate rainforest

Abstract Despite the ubiquitous presence of fungal endophytes in woody plants, mechanisms underlying variation in foliar fungal endophyte communities are poorly understood. Given that endophytes in woody plants are predominantly horizontally transmitted, fungal endophyte colonization of foliar tissues is likely to be influenced by plant resistance traits. Here, we evaluated the association between leaf resistance traits and colonization and community composition of horizontally transmitted endophytes (HTE) in 10 dominant trees species in a temperate rainforest in Southern Chile. Because resistance traits might restrict colonization of HTE, it was hypothesized that host trees with similar leaf resistance traits would show similar HTE communities. We measured leaf traits with reported roles in plant resistance against pathogens and/or herbivores: cell wall polysaccharides, leaf toughness, flavonoids, anthocyanins, terpenoids and chitinases. We also determined variation in fungal read counts, number of operational taxonomic units (OTUs), diversity and community structure across tree species. Foliar fungal endophyte communities were characterized using Illumina MiSeq sequencing of tagged amplicons of the ITS1 region. Fungal read counts were significantly different among host trees, while number of OTUs and diversity were similar among them. Multivariate analyses showed that community composition of endophytic fungi significantly differed among hosts, and that variation in cell wall, flavonoids, anthocyanins and terpenoids was associated with differences in HTE communities. We found that host trees sharing leaf resistance traits harbour similar HTE communities. Fungal read counts decreased with increasing content of cell wall polysaccharides, while endophyte diversity decreased with increasing anthocyanin levels. Fungal read counts, in contrast, were positively associated with terpenoids. Additionally, culture‐based data indicated that some HTE strains inhibited the growth of the common fungal pathogen Botrytis cinerea. Synthesis. Overall, we found that leaf resistance traits may influence fungal endophyte colonization and community composition in tree species, likely through deterrent effects of structural and chemical traits. We suggest that endophytic fungi that overcome resistance traits might play a role in plant protection.

Site-specific responses of foliar fungal microbiomes to nutrient addition and herbivory at different spatial scales.

The plant microbiome can affect host function in many ways and characterizing the ecological factors that shape endophytic (microbes living inside host plant tissues) community diversity is a key step in understanding the impacts of environmental change on these communities. Phylogenetic relatedness among members of a community offers a way of quantifying phylogenetic diversity of a community and can provide insight into the ecological factors that shape endophyte microbiomes. We examined the effects of experimental nutrient addition and herbivory exclusion on the phylogenetic diversity of foliar fungal endophyte communities of the grass species Andropogon gerardii at four sites in the Great Plains of the central USA. Using amplicon sequencing, we characterized the effects of fertilization and herbivory on fungal community phylogenetic diversity at spatial scales that spanned within‐host to between sites across the Great Plains. Despite increasing fungal diversity and richness, at larger spatial scales, fungal microbiomes were composed of taxa showing random phylogenetic associations. Phylogenetic diversity did not differ systematically when summed across increasing spatial scales from a few meters within plots to hundreds of kilometers among sites. We observed substantial shifts in composition across sites, demonstrating distinct but similarly diverse fungal communities were maintained within sites across the region. In contrast, at the scale of within leaves, fungal communities tended to be comprised of closely related taxa regardless of the environment, but there were no shifts in phylogenetic composition among communities. We also found that nutrient addition (fertilization) and herbivory have varying effects at different sites. These results suggest that the direction and magnitude of the outcomes of environmental modifications likely depend on the spatial scale considered, and can also be constrained by regional site differences in microbial diversity and composition.

Differentiating spatial from environmental effects on foliar fungal communities of Populus trichocarpa

AbstractAimFoliar fungi – pathogens, endophytes, epiphytes – form taxonomically diverse communities that affect plant health and productivity. The composition of foliar fungal communities is variable at spatial scales both small (e.g. individual plants) and large (e.g. continents), yet few studies have attempted to tease apart spatial from climatic factors influencing these communities. Moreover, few studies have sampled in more than 1 year to gauge interannual variation in community structure.LocationThe Pacific Northwest of western North America.TaxonFoliar fungi associated with the deciduous tree Populus trichocarpa.MethodsIn two consecutive years, we used DNA metabarcoding to characterize foliar fungal communities of Populus trichocarpa across its geographic range, which encompasses a sharp climatic transition as it crosses the Cascade Mountain Range. We used multivariate analyses to (a) test for and differentiate spatial and environmental factors affecting community composition and (b) test for temporal variation in community composition across spatial and environmental gradients.ResultsIn both study years, we found that foliar fungal community composition varied among sites and between regions (east vs. west of the Cascades). We found that climate explained more variation in community composition than geographic distance, although the majority of variation explained by each was shared. We also found that interannual variation in community composition depended on environmental context: communities located in the dry, eastern portion of the tree's geographic range varied more between study years than those located in the wet, western portion of the tree's range.Main conclusionsOur results suggest that the environment plays a greater role in structuring foliar fungal communities than dispersal limitation.

Differences in foliar endophyte communities of red alder (Alnus rubra) exposed to varying air pollutant levels

In the Pacific Northwest, Alnus rubra Bong. (red alder) is a common deciduous tree species especially prevalent in riparian corridors and disturbed sites, including metropolitan areas undergoing land use changes and development. Importantly, red alder is also considered a bioindicator for ozone pollution and, like all plants, harbors a diverse endophyte community that may interact with aerial pollutants. In this study, we surveyed foliar fungal endophyte communities (microfungi) in red alder leaves from the metropolitan area of Portland, Oregon, USA, using culture-based techniques, and found that communities differed significantly by site. Our results suggest that the fungal endophyte community composition in red alder leaves may be influenced in part by local air pollution sources, likely in conjunction with other site characteristics. As urban areas expand, more studies should focus on how the urban environment affects plant–microbe community ecology and endophyte–host interactions, as well as on the long-term consequences for other ecosystem processes such as leaf litter decomposition.

Foliar fungal endophyte communities are structured by environment but not host ecotype in Panicum virgatum (switchgrass).

Experimental tests of community assembly mechanisms for host-associated microbiomes in nature are lacking. Asymptomatic foliar fungal endophytes are a major component of the plant microbiome and are increasingly recognized for their impacts on plant performance, including pathogen defense, hormonal manipulation, and drought tolerance. However, it remains unclear whether fungal endophytes preferentially colonize certain host ecotypes or genotypes, reflecting some degree of biotic adaptation in the symbioses, or whether colonization is simply a function of spore type and abundance within the local environment. Whether host ecotype, local environment, or some combination of both controls the pattern of microbiome formation across hosts represents a new dimension to the age-old debate of nature versus nurture. Here, we used a reciprocal transplant design to explore the extent of host specificity and biotic adaptation in the plant microbiome, as evidenced by differential colonization of host genetic types by endophytes. Specifically, replicate plants from three locally-adapted ecotypes of the native grass Panicum virgatum (switchgrass) were transplanted at three geographically distinct field sites (one home and two away) in the Midwestern US. At the end of the growing season, plant leaves were harvested and the fungal microbiome characterized using culture-dependent sequencing techniques. Our results demonstrated that fungal endophyte community structure was determined by local environment (i.e., site), but not by host ecotype. Fungal richness and diversity also strongly differed by site, with lower fungal diversity at a riparian field site, whereas host ecotype had no effect. By contrast, there were significant differences in plant phenotypes across all ecotypes and sites, indicating ecotypic differentiation of host phenotype. Overall, our results indicate that environmental factors are the primary drivers of community structure in the switchgrass fungal microbiome.

Occurrence of common phyllosphere fungi of horse-chestnut (Aesculus hippocastanum) is unrelated to degree of damage by leafminer (Cameraria ohridella)

ABSTRACTThe horse chestnut leaf-miner (Cameraria ohridella Deschka & Dimić, Lepidoptera, Gracillariidae) is an invasive pest causing extensive damage to leaves of the horse-chestnut (Aesculus hippocastanum L.) in Europe. In Lithuania, C. ohridella invaded in 2002 causing wilting, browning and premature fall of A. hippocastanum leaves. The aim was to get a better understanding of possible linkages between foliar fungal communities and leaf-miner damage in A. hippocastanum. Leaves of A. hippocastanum, differentially damaged by C. ohridella, were collected in 10 sites in Lithuania. The fungal communities were described through DNA isolation and amplification using an ITS rRNA marker and Ion Torrent-sequencing. Clustering of 214,897 high-quality sequences resulted in 1017 non-singleton fungal taxa, among which Aureobasidium pullulans (28.2% of all fungal sequences), Endoconidioma populi (27.7%), Phoma fungicola (11.3%), Cladosporium ramotenellum (7.6%) and Cryptococcus sp. 2185_4 (5.0%) were most common. Correspondence analysis showed that fungal communities from heavily and slightly damaged leaves were largely intermingled, showing that in both types of samples fungal communities were similar. In conclusion, the study demonstrated that the phyllosphere of A. hippocastanum is inhabited by a high diversity of fungal species, the majority of which constitute generalist endophytes, epiphytes and saprotrophic fungi. The occurrence of common phyllosphere fungi was unrelated to the degree of damage by C. ohridella.

Persisting responses of salt marsh fungal communities to the Deepwater Horizon oil spill

The plant microbiome, composed of diverse interacting microorganisms, is thought to undergird host integrity and well-being. Though it is well understood that environmental perturbations like oil pollution can alter the diversity and composition of microbiomes, remarkably little is known about how disturbance alters plant-fungal associations. Using Next-Generation sequencing of the 18S rDNA internal transcribed spacer (ITS1) region, we examined outcomes of enduring oil exposure on aboveground leaf and belowground endophytic root and rhizosphere fungal communities of Spartina alterniflora, a highly valued ecosystem engineer in southeastern Louisiana marshes affected by the 2010 Deepwater Horizon accident. We found that aboveground foliar fungal communities exhibited site-dependent compositional turnover with consequent loss in diversity according to oiling history. Rhizosphere soil communities also exhibited shifts in community composition associated with oiling history, whereas root endophytic communities did not. Oiling did not increase or decrease similarities among aboveground and belowground communities within an individual host, indicating that host plant characteristics exert stronger control than external factors on fungal community composition. These results show that fungal community responses to oiling vary within tissues of the same host plant, and that differences in the local environment, or alternatively, site-specific differences in residual oil constrain the magnitude of exposure responses. Our study offers novel perspectives on how environmental contaminants and perturbations can influence plant microbiomes, highlighting the importance of assessing long-term ecological outcomes of oil pollution to better understand how shifts in microbial communities influence plant performance and ecosystem function. Our findings are relevant to coastal management programs tasked with responding to oil spills and increasing pressures arising from intensifying development and climate change. Understanding how modification of plant-microbiome associations influences plant performance, particularly of ecosystem engineers like S. alterniflora, can help guide efforts to protect and restore at-risk coastal ecosystems.

Knowing your neighbourhood-the effects of Epichloë endophytes on foliar fungal assemblages in perennial ryegrass in dependence of season and land-use intensity.

Epichloë endophytes associated with cool-season grass species can protect their hosts from herbivory and can suppress mycorrhizal colonization of the hosts’ roots. However, little is known about whether or not Epichloë endophyte infection can also change the foliar fungal assemblages of the host. We tested 52 grassland study sites along a land-use intensity gradient in three study regions over two seasons (spring vs. summer) to determine whether Epichloë infection of the host grass Lolium perenne changes the fungal community structure in leaves. Foliar fungal communities were assessed by Next Generation Sequencing of the ITS rRNA gene region. Fungal community structure was strongly affected by study region and season in our study, while land-use intensity and infection with Epichloë endophytes had no significant effects. We conclude that effects on non-systemic endophytes resulting from land use practices and Epichloë infection reported in other studies were masked by local and seasonal variability in this study’s grassland sites.

Whitebark pine foliar fungal endophyte communities in the southern Cascade Range, USA: Host mycobiomes and white pine blister rust

Abstract We present an assessment of relationships between correlates, signs, and symptoms of white pine blister rust and fungal endophyte communities of whitebark pine (Pinus albicaulis) foliage in the southern Cascade Range, OR, USA. Parsimonious combinations of historically important predictors of blister rust provided effective logistic models (AUC = 0.83) for predicting blister rust infection in whitebark pines at our sites. Holding ten other predictors constant, multivariate endophyte community structure varied significantly with host tree diameter, mountain aspect, the latitudinal coordinates at which samples were collected, and proximal alternative host plants of the white pine blister rust pathogen. After controlling for multiple comparisons, mountain aspect remained a significant source of variation in endophyte community composition even at the taxonomic rank of class, while the presence/absence of blister rust cankers did not remain significant. We discuss overarching mechanisms that may contribute to novel trends observed in mycobiome variation.

A heritable symbiont and host‐associated factors shape fungal endophyte communities across spatial scales

Abstract Microbial ecologists are intensely interested in the processes governing microbial community assembly; however, progress has been limited by a lack of studies that span multiple geographical scales and levels of biological organization. We used high throughput sequencing to characterize foliar fungal endophyte communities and host plant genetic structure both within and among 24 populations of spotted locoweed Astragalus lentiginosus across the Great Basin Desert. Across the Great Basin, both within and among populations of the host plant, fungal endophyte richness was predicted by plant size and variation in the seed‐borne, heritable fungus, Alternaria fulva, which produces the bioactive alkaloid swainsonine. The degree of between‐plant turnover in the endophyte community was inversely related to host plant inbreeding and average plant size and positively related to the relative abundance of A. fulva. Plant size was inversely related to endophyte community richness, both among and within populations. The genetic and physical distance between host populations was not predictive of differences in fungal community structure. Synthesis. Through pairing intensive local and regional sampling, we uncovered a primacy of deterministic forces imposed by a heritable symbiont on the community structure of locoweed endophytes.

Distributions of ectomycorrhizal and foliar endophytic fungal communities associated with Pinus ponderosa along a spatially constrained elevation gradient.

Understanding distributions of plant-symbiotic fungi is important for projecting responses to environmental change. Many coniferous trees host ectomycorrhizal fungi (EM) in association with roots and foliar endophytic fungi (FE) in leaves. We examined how EM and FE associated with Pinus ponderosa each vary in abundance, diversity, and community structure over a spatially constrained elevation gradient that traverses four plant communities, 4°C in mean annual temperature, and 15 cm in mean annual precipitation. We sampled 63 individuals of Pinus ponderosa in 10 sites along a 635 m elevation gradient that encompassed a geographic distance of 9.8 km. We used standard methods to characterize each fungal group (amplified and sequenced EM from root tips; isolated and sequenced FE from leaves). Abundance and diversity of EM were similar across sites, but community composition and distributions of the most common EM differed with elevation (i.e., with climate, soil chemistry, and plant communities). Abundance and composition of FE did not differ with elevation, but diversity peaked in mid-to-high elevations. Our results suggest relatively tight linkages between EM and climate, soil chemistry, and plant communities. That FE appear less linked with these factors may speak to limitations of a culture-based approach, but more likely reflects the small spatial scale encompassed by our study. Future work should consider comparable methods for characterizing these functional groups, and additional transects to understand relationships of EM and FE to environmental factors that are likely to shift as a function of climate change.

Foliar fungi of Betula pendula: impact of tree species mixtures and assessment methods

Foliar fungi of silver birch (Betula pendula) in an experimental Finnish forest were investigated across a gradient of tree species richness using molecular high-throughput sequencing and visual macroscopic assessment. We hypothesized that the molecular approach detects more fungal taxa than visual assessment, and that there is a relationship among the most common fungal taxa detected by both techniques. Furthermore, we hypothesized that the fungal community composition, diversity, and distribution patterns are affected by changes in tree diversity. Sequencing revealed greater diversity of fungi on birch leaves than the visual assessment method. One species showed a linear relationship between the methods. Species-specific variation in fungal community composition could be partially explained by tree diversity, though overall fungal diversity was not affected by tree diversity. Analysis of specific fungal taxa indicated tree diversity effects at the local neighbourhood scale, where the proportion of birch among neighbouring trees varied, but not at the plot scale. In conclusion, both methods may be used to determine tree diversity effects on the foliar fungal community. However, high-throughput sequencing provided higher resolution of the fungal community, while the visual macroscopic assessment detected functionally active fungal species.

Canopy tree species and openness affect foliar endophytic fungal communities of understory seedlings

AbstractThere is growing evidence demonstrating the diversity of foliar endophytic fungi and their ecological roles in the survival of tree seedlings. However, the factors that shape fungal communities in tree seedlings within natural forest ecosystems remain poorly understood. Here, we evaluated the composition of foliar endophytic fungi growing in current‐year seedlings of Cornus controversa and Prunus grayana in a cool temperate deciduous forest through a seed‐sowing experiment and fungal isolation. The composition of endophytic fungi was affected by canopy tree species, canopy openness, and time after germination. In total, 27 and 22 fungal taxa were isolated from C. controversa and P. grayana seedlings, respectively. The dominant fungal taxa in both seedling species were Colletotorichum spp., and their isolation frequencies were higher under C. controversa canopies than under P. grayana canopies; the frequencies also increased with time after germination. These results suggest that overstory tree species strongly influences the endophytic fungal communities of understory seedlings.

Vertical stratification of the foliar fungal community in the world's tallest trees.

The aboveground tissues of plants host numerous, ecologically important fungi, yet patterns in the spatial distribution of these fungi remain little known. Forest canopies in particular are vast reservoirs of fungal diversity, but intracrown variation in fungal communities has rarely been explored. Knowledge of how fungi are distributed throughout tree crowns will contribute to our understanding of interactions between fungi and their host trees and is a first step toward investigating drivers of community assembly for plant-associated fungi. Here we describe spatial patterns in fungal diversity within crowns of the world's tallest trees, coast redwoods (Sequoia sempervirens). We took a culture-independent approach, using the Illumina MiSeq platform, to characterize the fungal assemblage at multiple heights within the crown across the geographical range of the coast redwood. Within each tree surveyed, we uncovered evidence for vertical stratification in the fungal community; different portions of the tree crown harbored different assemblages of fungi. We also report between-tree variation in the fungal community within redwoods. Our results suggest the potential for vertical stratification of fungal communities in the crowns of other tall tree species and should prompt future study of the factors giving rise to this stratification.

Foliar fungal communities strongly differ between habitat patches in a landscape mosaic.

BackgroundDispersal events between habitat patches in a landscape mosaic can structure ecological communities and influence the functioning of agrosystems. Here we investigated whether short-distance dispersal events between vineyard and forest patches shape foliar fungal communities. We hypothesized that these communities homogenize between habitats over the course of the growing season, particularly along habitat edges, because of aerial dispersal of spores.MethodsWe monitored the richness and composition of foliar and airborne fungal communities over the season, along transects perpendicular to edges between vineyard and forest patches, using Illumina sequencing of the Internal Transcribed Spacer 2 (ITS2) region.ResultsIn contrast to our expectation, foliar fungal communities in vineyards and forest patches increasingly differentiate over the growing season, even along habitat edges. Moreover, the richness of foliar fungal communities in grapevine drastically decreased over the growing season, in contrast to that of forest trees. The composition of airborne communities did not differ between habitats. The composition of oak foliar fungal communities change between forest edge and centre.DiscussionThese results suggest that dispersal events between habitat patches are not major drivers of foliar fungal communities at the landscape scale. Selective pressures exerted in each habitat by the host plant, the microclimate and the agricultural practices play a greater role, and might account for the differentiation of foliar fugal communities between habitats.

Do foliar fungal communities of Norway spruce shift along a tree species diversity gradient in mature European forests?

Abstract Foliar fungal species are diverse and colonize all plants, though whether forest tree species composition influences the distribution of these fungal communities remains unclear. Fungal communities include quiescent taxa and the functionally important and metabolically active taxa that respond to changes in the environment. To determine fungal community shifts along a tree species diversity gradient, needles of Norway spruce were sampled from trees from four mature European forests. We hypothesized that the fungal communities and specific fungal taxa would correlate with tree species diversity. Furthermore, the active fungal community, and not the total community, would shift along the tree diversity gradient. High-throughput sequencing showed significant differences in the fungal communities in the different forests, and in one forest, tree diversity effects were observed, though this was not a general phenomenon. Our study also suggests that studying the metabolically active community may not provide additional information about community composition or diversity.

Deciphering the Pathobiome: Intra- and Interkingdom Interactions Involving the Pathogen Erysiphe alphitoides.

Plant-inhabiting microorganisms interact directly with each other, forming complex microbial interaction networks. These interactions can either prevent or facilitate the establishment of new microbial species, such as a pathogen infecting the plant. Here, our aim was to identify the most likely interactions between Erysiphe alphitoides, the causal agent of oak powdery mildew, and other foliar microorganisms of pedunculate oak (Quercus robur L.). We combined metabarcoding techniques and a Bayesian method of network inference to decipher these interactions. Our results indicate that infection with E. alphitoides is accompanied by significant changes in the composition of the foliar fungal and bacterial communities. They also highlight 13 fungal operational taxonomic units (OTUs) and 13 bacterial OTUs likely to interact directly with E. alphitoides. Half of these OTUs, including the fungal endophytes Mycosphaerella punctiformis and Monochaetia kansensis, could be antagonists of E. alphitoides according to the inferred microbial network. Further studies will be required to validate these potential interactions experimentally. Overall, we showed that a combination of metabarcoding and network inference, by highlighting potential antagonists of pathogen species, could potentially improve the biological control of plant diseases.

Friend or foe? Biological and ecological traits of the European ash dieback pathogen Hymenoscyphus fraxineus in its native environment.

Hymenoscyphus fraxineus, an introduced ascomycete fungus and primary causal agent of European ash dieback, was investigated on Fraxinus mandshurica trees in its native range in Primorye region of Far East Russia. This evidence is the first report of H. fraxineus on healthy, asymptomatic F. mandshurica trees. High-throughput sequencing revealed 49 distinct fungal taxa associated with leaves of F. mandshurica, 12 of which were identified to species level. Phyllosphere fungal assemblages were similar among sites despite being largely geographically distant. Many organisms comprising the foliar fungal community on F. mandshurica in Far East Russia have similarity to those reported inhabiting F. excelsior in Europe based on previous studies. However, Mycosphaerella sp., the most dominant species in this study and detected in nearly all samples, was associated only with F. mandshurica. Genetic diversity of H. fraxineus was significantly higher in the Far East Russian population than in Europe. In contrast to its aggressive behaviour on Fraxinus excelsior in Europe, H. fraxineus appears to be a benign associate of indigenous F. mandshurica that initially induces quiescent and asymptomatic infections in healthy trees prior to active host colonization normally associated with modification of host tissue during senescence.

A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda)

Fungal endophytes represent one of the most ubiquitous plant symbionts on Earth and are phylogenetically diverse. The structure and diversity of endophyte communities have been shown to depend on host taxa and climate, but there have been relatively few studies exploring endophyte communities throughout host maturity. We compared foliar fungal endophyte communities between seedlings and adult trees of loblolly pines (Pinus taeda) at the same seasons and locations by culturing and culture-independent methods. We sequenced the internal transcribed spacer region and adjacent partial large subunit nuclear ribosomal RNA gene (ITS–LSU amplicon) to delimit operational taxonomic units and phylogenetically characterize the communities. Despite the lower infection frequency in seedlings compared to adult trees, seedling needles were receptive to a more diverse community of fungal endophytes. Culture-free method confirmed the presence of commonly cultured OTUs from adult needles but revealed several new OTUs from seedling needles that were not found with culturing methods. The two most commonly cultured OTUs in adults were rarely cultured from seedlings, suggesting that host age is correlated with a selective enrichment for specific endophytes. This shift in endophyte species dominance may be indicative of a functional change between these fungi and their loblolly pine hosts.

Correlation analysis between foliar endophytic fungi of Salvia miltiorrhiza and effective components

The aim of this study was to comprehensively investigate the correlations between foliar fungal endophyte communities and effective components accumulations in Salvia miltiorrhiza. Foliar samples of S. miltiorrhiza were collected in 5 different areas. Their fungal endophyte communities and effective component contents were determined by denaturing gradient gel electrophoresis (DGGE) and high performance liquid chromatography (HPLC), respectively. The results showed that, for characteristics of foliar fungal endophyte communities and effective component contents, there were both similarities and differences among the five samples. Correlation analysis of DGGEs' band and 24 effective components revealed a significant correlations (P < 0.01). For examples, 4 bands (15, 18, 23 and 26) were all significantly correlated with the accumulations of caffeic acid, salvianolic acid B, salvianolic acid C and dihydrotanshinone I.