Community Analysis Reveals Close Affinities Between Endophytic and Endolichenic Fungi in Mosses and Lichens

Endolichenic fungi live in close association with algal photobionts inside asymptomatic lichen thalli and resemble fungal endophytes of plants in terms of taxonomy, diversity, transmission mode, and evolutionary history. This similarity has led to uncertainty regarding the distinctiveness of endolichenic fungi compared with endophytes. Here, we evaluate whether these fungi represent distinct ecological guilds or a single guild of flexible symbiotrophs capable of colonizing plants or lichens indiscriminately. Culturable fungi were sampled exhaustively from replicate sets of phylogenetically diverse plants and lichens in three microsites in a montane forest in southeastern Arizona (USA). Intensive sampling combined with a small spatial scale permitted us to decouple spatial heterogeneity from host association and to sample communities from living leaves, dead leaves, and lichen thalli to statistical completion. Characterization using data from the nuclear ribosomal internal transcribed spacer and partial large subunit (ITS-LSU rDNA) provided a first estimation of host and substrate use for 960 isolates representing five classes and approximately 16 orders, 32 families, and 65 genera of Pezizomycotina. We found that fungal communities differ at a broad taxonomic level as a function of the phylogenetic placement of their plant or lichen hosts. Endolichenic fungal assemblages differed as a function of lichen taxonomy, rather than substrate, growth form, or photobiont. In plants, fungal communities were structured more by plant lineage than by the living vs. senescent status of the leaf. We found no evidence that endolichenic fungi are saprotrophic fungi that have been “entrapped” by lichen thalli. Instead, our study reveals the distinctiveness of endolichenic communities relative to those in living and dead plant tissues, with one notable exception: we identify, for the first time, an ecologically flexible group of symbionts that occurs both as endolichenic fungi and as endophytes of mosses.     

Host and geographic structure of endophytic and endolichenic fungi at a continental scale

? Premise of the study: Endophytic and endolichenic fungi occur in healthy tissues of plants and lichens, respectively, playing potentially important roles in the ecology and evolution of their hosts. However, previous sampling has not comprehensively evaluated the biotic, biogeographic, and abiotic factors that structure their communities. ? Methods: Using molecular data we examined the diversity, composition, and distributions of 4154 endophytic and endolichenic Ascomycota cultured from replicate surveys of ca. 20 plant and lichen species in each of five North American sites (Madrean coniferous forest, Arizona; montane semideciduous forest, North Carolina; scrub forest, Florida; Beringian tundra and forest, western Alaska; subalpine tundra, eastern central Alaska). ? Key results: Endolichenic fungi were more abundant and diverse per host species than endophytes, but communities of endophytes were more diverse overall, reflecting high diversity in mosses and lycophytes. Endophytes of vascular plants were largely distinct from fungal communities that inhabit mosses and lichens. Fungi from closely related hosts from different regions were similar in higher taxonomy, but differed at shallow taxonomic levels. These differences reflected climate factors more strongly than geographic distance alone. ? Conclusions: Our study provides a first evaluation of endophytic and endolichenic fungal associations with their hosts at a continental scale. Both plants and lichens harbor abundant and diverse fungal communities whose incidence, diversity, and composition reflect the interplay of climatic patterns, geographic separation, host type, and host lineage. Although culture-free methods will inform future work, our study sets the stage for empirical assessments of ecological specificity, metabolic capability, and comparative genomics.     

Fungal communities living within leaves of native Hawaiian dicots are structured by landscape‐scale variables as well as by host plants

A phylogenetically diverse array of fungi live within healthy leaf tissue of dicotyledonous plants. Many studies have examined these endophytes within a single plant species and/or at small spatial scales, but landscape‐scale variables that determine their community composition are not well understood, either across geographic space, across climatic conditions, or in the context of host plant phylogeny. Here, we evaluate the contributions of these variables to endophyte beta diversity using a survey of foliar endophytic fungi in native Hawaiian dicots sampled across the Hawaiian archipelago. We used Illumina technology to sequence fungal ITS1 amplicons to characterize foliar endophyte communities across five islands and 80 host plant genera. We found that communities of foliar endophytic fungi showed strong geographic structuring between distances of 7 and 36 km. Endophyte community structure was most strongly associated with host plant phylogeny and evapotranspiration, and was also significantly associated with NDVI, elevation and solar radiation. Additionally, our bipartite network analysis revealed that the five islands we sampled each harboured significantly specialized endophyte communities. These results demonstrate how the interaction of factors at large and small spatial and phylogenetic scales shapes fungal symbiont communities.     

Diversity,community composition,and bioactivity of cultivable fungal endophytes in saline and dry soils in deserts

Fungal endophyte communities are poorly investigated in extreme habitats such as deserts. We used cultivation and Sanger sequencing to investigate the effects of environmental variables on the endophytic fungal communities of eight Iranian desert plants. Host species was the main factor shaping the endophyte composition, while soil type additionally affected endophytes of above- and below-ground organs. Redundancy analysis showed that soil pH and electric conductivity determine fungal endophyte communities in plants in dry and saline soils. In a follow-up experiment, we showed that these endophytes could be used in crop production under salinity/drought stress and as biocontrol agents. Although compared to other ecosystems, the endophytic fungi associated with the studied Iranian desert plants are of low diversity, our results suggest that they probably play an essential role in the survival of their hosts. Further investigation is necessary to evaluate the potential benefits and applicability of such endophytes in agricultural practices in drylands.     

Host associations and beta diversity of fungal endophyte communities in New Guinea rainforest trees

Processes shaping the distribution of foliar fungal endophyte species remain poorly understood. Despite increasing evidence that these cryptic fungal symbionts of plants mediate interactions with pathogens and herbivores, there remain basic questions regarding the extent to which dispersal limitation and host specificity might shape fungal endophyte community composition in rainforests. To assess the relative importance of spatial pattern and host specificity, we isolated fungi from a sample of mapped trees in lowland Papua New Guinea. Sequences of the internal transcribed spacer (ITS) region were obtained for 2079 fungal endophytes from three sites and clustered into molecular operational taxonomic units (MOTUs) at 95% similarity. Multivariate analyses suggest that host affinity plays a significant role in structuring endophyte community composition whereas there was no evidence of endophyte spatial pattern at the scale of tens to hundreds of metres. Differences in endophyte communities between sampled trees were weakly correlated with variation in foliar traits but not with tree species relatedness. The dominance of relatively few generalist endophytes and the presence of a large number of rare MOTUs was a consistent observation at three sites separated by hundreds of kilometres and regional turnover was low. Host specificity appears to play a relatively weak but more important role than dispersal limitation in shaping the distribution of fungal endophyte communities in New Guinea forests. Our results suggest that in the absence of strong ecological gradients and host turnover, beta diversity of endophyte communities could be low in large areas of contiguous forest.     

Biogeographic and phylogenetic patterns in diversity of liverwort-associated endophytes

Liverworts harbor diverse fungi, including endophytes, in their healthy tissues. To address whether patterns of endophyte diversity are correlated with host phylogeny or geography, we designed a broad geographic survey with controlled phylogenetic host sampling. We collected liverworts in North Carolina, Washington, Idaho, British Columbia, Germany, and New Zealand and identified endophytes using culture-based and molecular methods. Of the major lineages of filamentous ascomycetes recovered, 53-88% belonged to the Xylariales. Endophyte accumulation curves did not saturate, and singleton sequences were dominant in each region, suggesting that liverwort endophyte communities are diverse. There was no significant difference in species richness between regional endophyte communities; however, total richness estimators indicated that North Carolina and New Zealand have richer communities than do Germany and the Pacific Northwest. This pattern reflects lower per-host endophyte density and prevalence of a common, shared sequence group in Germany and the Pacific Northwest. Although species richness was relatively low in the Pacific Northwest, the greatest phylogenetic diversity of endophytes was recovered there. Tests for regional and host specificity revealed that endophyte floras of hosts within a geographic area are more similar to one another than to those of closely related hosts. Geographic distance, not host phylogeny, best explains differences among communities.     

广义与狭义植物内生生物的定义及其研究进展

本文从比较植物内生生物的概念入手,提出了广义与狭义两种植物内生生物的定义;归纳了植物内生生物的物种多样性、分布、及其代谢特点;分析了植物内生生物与寄主植物的关系;探讨了部分植物内生生物潜在的价值和应用前景。     

Endophytic fungal communities in woody perennials of three tropical forest types of the Western Ghats,southern India

Fungal endophytes of tropical trees are expected to be exceptionally species rich as a consequence of high tree diversity in the tropics and the purported host restriction among the endophytes. Based on this premise, endophytes have been regarded as a focal group for estimating fungal numbers because their possible hyperdiverse nature would reflect significantly global fungal diversity. We present our consolidated ten-year work on 75 dicotyledonous tree hosts belonging to 33 families and growing in three different types of tropical forests of the NBR in the Western Ghats, southern India. We conclude that endophyte diversity in these forests is limited due to loose host affiliations among endophytes. Some endophytes have a wide host range and colonize taxonomically disparate hosts suggesting adaptations in them to counter a variety of defense chemicals in their hosts. Furthermore, such polyphagous endophytes dominate the endophyte assemblages of different tree hosts. Individual leaves may be densely colonized but only by a few endophyte species. It appears that the environment (the type of forest in this case) has a larger role in determining the endophyte assemblage of a plant host than the taxonomy of the host plant. Thus, different tropical plant communities have to be studied for their endophyte diversity to test the generalization that endophytes are hyperdiverse in the tropics, estimate their true species richness, and use them as a predictor group for more accurate assessment of global fungal diversity.     

庞泉沟自然保护区常见禾本科植物内生真菌分布及其影响因素

以庞泉沟自然保护区4种不同群落类型林地和灌丛下的优势禾草为研究对象,调查不同禾草内生真菌感染率,研究内生真菌感染的禾草种群与原生生境的土壤理化性质、土壤酶活性及土壤微生物群落结构的相互关系。结果发现,影响紫羊茅、中亚薹草和野燕麦染菌率的主要因子是土壤水分含量和碳氮比,且土壤C、N、S以及土壤水分含量与紫羊茅的内生真菌染菌率成正相关,土壤蔗糖酶、脲酶和酸性磷酸酶与紫羊茅的感染率显著正相关,但中亚薹草与紫羊茅的结果相反。丝盖伞科是华北落叶松林和油松林土壤微生物群落的优势真菌,但不同染菌植被群落类型下的优势细菌微生物群落组成不同。总体看来,4种森林群落类型下禾草内生真菌染菌率与土壤理化性质相关性不同,且受到的生态因子影响大小也有差异,内生真菌感染导致各植被群落类型的土壤微生物群落结构具有差异,其优势土壤真菌和土壤细菌组成不同,这有助于进一步认识原生生境下不同感染率的禾草种群对土壤微生物群落结构的影响及其在生态系统中的各异生态学功能。     

Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees

Understanding how fungal endophyte communities differ in abundance, diversity, taxonomic composition, and host affinity over the geographic ranges of their hosts is key to understanding the ecology and evolutionary context of endophyte–plant associations. We examined endophytes associated with healthy photosynthetic tissues of three closely related tree species in the Cupressaceae (Coniferales): two native species within their natural ranges [Juniperus virginiana in a mesic semideciduous forest, North Carolina (NC); Cupressus arizonica, under xeric conditions, Arizona (AZ)], and a non-native species planted in each site (Platycladus orientalis). Endophytes were recovered from 229 of 960 tissue segments and represented at least 35 species of Ascomycota. Isolation frequency was more than threefold greater for plants in NC than in AZ, and was 2.5 (AZ) to four (NC) times greater for non-native Platycladus than for Cupressus or Juniperus. Analyses of ITS rDNA for 109 representative isolates showed that endophyte diversity was more than twofold greater in NC than in AZ, and that endophytes recovered in AZ were more likely to be host-generalists relative to those in NC. Different endophyte genera dominated the assemblages of each host species/locality combination, but in both localities, Platycladus harboured less diverse and more cosmopolitan endophytes than did either native host. Parsimony and Bayesian analyses for four classes of Ascomycota (Dothideomycetes, Sordariomycetes, Pezizomycetes, Eurotiomycetes) based on LSU rDNA data (ca 1.2 kb) showed that well-supported clades of endophytes frequently contained representatives of a single locality or host species, underscoring the importance of both geography and host identity in shaping a given plant's endophyte community. Together, our data show that not only do the abundance, diversity, and taxonomic composition of endophyte communities differ as a function of host identity and locality, but that host affinities of those communities are variable as well.     

Endophyte research: going beyond isolation and metabolite documentation

Many fungi belonging to mostly Ascomycota inhabit living tissues of plants of all major lineages without causing any visible symptoms. Termed horizontally transmitted endophytes, they have been investigated mostly for their capacity to produce bioactive secondary metabolites. However, many questions regarding the interactions between endophytes and their plant hosts, phytophagous insects and other fungi remain unanswered. This review highlights some of these areas of endophyte biology about which very little or no knowledge exists. Information garnered' using modern methodologies' on these grey areas of ‘endophytism’ (endophytic mode of lifestyle) would help immensely in understanding the evolution of endophytes of aerial plant tissues and in exploiting endophytes in various fields of biotechnology.     

Non-systemic fungal endophytes of grasses

Many fungi behave as endophytes in grasses. Unlike the well known Epichloë/Neotyphodium species, most other endophytes are not capable of systemic colonization of plant organs, or seed transmission. The species diversity of the non-systemic endophytic mycobiota of grasses is large, dominated by ascomycetes. The relative abundance of species is very unequal, a few dominant taxa like Acremonium, Alternaria, Cladosporium, Epicoccum and Penicillium spp., occur in many grasses and locations. In contrast, many rare species are isolated only once in endophyte surveys. The possible ecological functions of endophytes are diverse, and often unknown. Latent pathogens represent a small fraction of endophytic mycobiotas, indicating that many non-pathogenic fungal taxa are able to enter plants overriding defence reactions. Some dominant species behave as latent saprotrophs, sporulating when the host tissue dies. Endofungal viruses and bacteria occur among endophytic species, but their effect in their hosts is largely unknown.     

Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic,medicinal orchid (<Emphasis Type="Italic">Dendrobium nobile</Emphasis>): estimation and characterization

Although the terrestrial and temperate orchids–fungal biology have been largely explored, knowledge of tropical epiphytic orchids–fungus relationships, especially on the ecological roles imparted by non-mycorrhizal fungal endophytes, is less known. Exploitation of the endophytic fungal mycobiota residing in epiphytic orchid plants may be of great importance to further elucidate the fungal ecology in this special habitat as well as developing new approaches for orchid conversations. The composition of fungal endophytes associated with leaves, stems and roots of an epiphytic orchid (Dendrobium nobile), a famous Chinese traditional medicinal plant, was investigated. Microscopic imaging, culture-dependant method and molecular phylogeny were used to estimate their entity and diversity. Totally, there were 172 isolates, at least 14 fungal genera and 33 different morphospecies recovered from 288 samples. Ascomycetes, coelomycetes and hyphomycetes were three major fungal groups. There were higher overall colonization and isolation rates of endophytic fungi from leaves than from other tissues. Guignardia mangiferae was the dominant fungal species within leaves; while the endophytic Xylariaceae were frequently observed in all plant tissues; Colletotrichum, Phomopsis and Fusarium were also frequently observed. Phylogenetic analysis based on ITS gene revealed the high diversity of Xylariacea fungi and relatively diverse of non-Xylariacea fungi. Some potentially promising beneficial fungi such as Clonostachys rosea and Trichoderma chlorosporum were found in roots. This is the first report concerning above-ground and below-ground endophytic fungi community of an epiphytic medicinal orchid, suggesting the ubiquitous distribution of non-mycorrhizal fungal endophytes in orchid plants together with heterogeneity and tissue specificity of the endophyte assemblage. Possible physiological functions played by these fungal endophytes and their potential applications are also discussed briefly. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

禾本科植物内生真菌研究 5.中国产部分epichloё内生真菌的遗传多样性

Epichloё内生真菌(epichloё endophytes)是禾本科植物的共生真菌,开展其多样性研究对菌株的生物学特性和进化规律的了解以及开发利用都有着十分重要的意义。本研究筛选了12条随机引物对24株epichloё内生真菌进行RAPD扩增,探讨真菌种类、宿主种类、采集地和菌株间的遗传多样性。结果表明,宿主为拂子茅Calamagrostis sp.、雀麦Bromus sp.、披碱草Elymus sp.、小颖羊茅Festuca parvigluma Steud.的菌株分别独立聚为一个分枝;而宿主为鹅观草Roegneria spp.的Neotyphodium属菌株聚类情况较为复杂;宿主为早熟禾Poas pp.的菌株分别在三个不同的分枝,表现出丰富的遗传多样性。以上结果反应了我国具有丰富的epichloё内生真菌资源,它们和其宿主之间有着比较明显的相互关系。本研究还显示,采集于南京的拂子茅与其内生真菌共生可能发生在较久远的年代。     

Aphid genotypes vary in their response to the presence of fungal endosymbionts in host plants

Genetic variation for fitness‐relevant traits may be maintained in natural populations by fitness differences that depend on environmental conditions. For herbivores, plant quality and variation in chemical plant defences can maintain genetic variation in performance. Apart from plant secondary compounds, symbiosis between plants and endosymbiotic fungi (endophytes) can produce herbivore‐toxic compounds. We show that there is significant variation among aphid genotypes in response to endophytes by comparing life‐history traits of 37 clones of the bird cherry‐oat aphid Rhopalosiphum padi feeding on endophyte‐free and endophyte‐infected tall fescue Lolium arundinaceum. Clonal variation for life‐history traits was large, and most clones performed better on endophyte‐free plants. However, the clones differed in the relative performance across the two environments, resulting in significant genotype × environment interactions for all reproductive traits. These findings suggest that natural variation in prevalence of endophyte infection can contribute to the maintenance of genetic diversity in aphid populations.     

Fungal endophyte communities in two tropical forests of southern India: diversity and host affiliation

Fifteen tree species from a tropical dry thorn forest and fifteen tree species from a tropical dry deciduous forest in the Mudumalai Wildlife Sanctuary, Nilgiri Biosphere Reserve, southern India, were surveyed for their foliar endophyte communities during the dry and wet seasons. Surface sterilized leaf segments of uniform dimension were plated on nutrient agar and culturable endophytes growing from the segments were identified. Endophyte diversity was greater in the dry thorn forest than in the dry deciduous forest in the dry season. Although the isolation frequency of culturable endophytes increased for both forests during the wet season, the assemblages were represented not by any unique fungal species but by the commonly occurring ones. Furthermore, although individual leaves were densely colonized by endophytes, only a few species of endophytes colonized the whole leaves; and, only a few fungal species dominated the foliar endophytic communities and were common for both forests during both dry and wet seasons. Thus, even under wet conditions that favour dispersal and infection by fungi, the endophyte diversity increased only marginally, an indication that certain tropical forests are not hyperdiverse with reference to fungal endophytes. This should be considered when using culturable endophyte diversity as a surrogate for estimating global fungal diversity.     

Interactions between Fusarium verticillioides, Ustilago maydis, and Zea mays: an endophyte, a pathogen, and their shared plant host

Highly diverse communities of microbial symbionts occupy eukaryotic organisms, including plants. While many well-studied symbionts may be characterized as either parasites or as mutualists, the prevalent but cryptic endophytic fungi are less easily qualified because they do not cause observable symptoms of their presence within their host. Here, we investigate the interactions of an endophytic fungus, Fusarium verticillioides with a pathogen, Ustilago maydis, as they occur within maize (Zea mays). We used experimental inoculations to evaluate metabolic mechanisms by which these three organisms might interact. We assessed the impacts of fungal-fungal interactions on endophyte and pathogen growth within the plant, and on plant growth. We find that F. verticillioides modulates the growth of U. maydis and thus decreases the pathogen's aggressiveness toward the plant. With co-inoculation of the endophyte with the pathogen, plant growth is similar to that which would be gained without the pathogen present. However, the endophyte may also break down plant compounds that limit U. maydis growth, and obtains a growth benefit from the presence of the pathogen. Thus, an endophyte such as F. verticillioides may function as both a defensive mutualist and a parasite, and express nutritional modes that depend on ecological context.     

Fungal Epiphytes and Endophytes of Coffee Leaves (Coffea arabica)

Plants harbor diverse communities of fungi and other microorganisms. Fungi are known to occur both on plant surfaces (epiphytes) and inside plant tissues (endophytes), but the two communities have rarely been compared. We compared epiphytic and endophytic fungal communities associated with leaves of coffee (Coffea arabica) in Puerto Rico. We asked whether the dominant fungi are the same in both communities, whether endophyte and epiphyte communities are equally diverse, and whether epiphytes and endophytes exhibit similar patterns of spatial heterogeneity among sites. Leaves of naturalized coffee plants were collected from six sites in Puerto Rico. Epiphytic and endophytic fungi were isolated by placing leaf pieces on potato dextrose agar without and with surface sterilization, respectively. A total of 821 colonies were isolated and grouped into 131 morphospecies. The taxonomic affinities of the four most common nonsporulating fungi were determined by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region: two grouped with Xylaria and one each with Botryosphaeria and Guignardia. Of the most common genera, Pestalotia and Botryosphaeria were significantly more common as epiphytes; Colletotrichum, Xylaria, and Guignardia were significantly more common as endophytes. Suprisingly, more morphospecies occurred as endophytes than as epiphytes. Differences among sites in number of fungi per plant were significant. Thus epiphytic and endophytic communities differed greatly on a single leaf, despite living only millimeters apart, and both communities differed from site to site. Significant correlations between occurrence of fungal morphospecies suggested that fungi may have positive or negative effects on their neighbors. This is the first quantitative comparison of epiphytic and endophytic fungal floras in any plant, and the first to examine endophytic fungi or epiphytic fungi in leaves of coffee, one of the world’s most valuable crops.