Effects of host plant environment and Ustilago maydis infection on the fungal endophyte community of maize (Zea mays)

The focus of many fungal endophyte studies has been how plants benefit from endophyte infection. Few studies have investigated the role of the host plant as an environment in shaping endophyte community diversity and composition. The effects that different attributes of the host plant, that is, host genetic variation, host variation in resistance to the fungal pathogen Ustilago maydis and U. maydis infection, have on the fungal endophyte communities in maize (Zea mays) was examined. The internal transcribed spacer (ITS) region of the rDNA was sequenced to identify fungi and the endophyte communities were compared in six maize lines that varied in their resistance to U. maydis. It was found that host genetic variation, as determined by maize line, had significant effects on species richness, while the interactions between line and U. maydis infection and line and field plot had significant effects on endophyte community composition. However, the effects of maize line were not dependent on whether lines were resistant or susceptible to U. maydis. Almost 3000 clones obtained from 58 plants were sequenced to characterize the maize endophyte community. These results suggest that the endophyte community is shaped by complex interactions and factors, such as inoculum pool and microclimate, may be important.     

Endophyte microbiome diversity in micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities.     

Response of fungal endophyte communities within Andropogon gerardii (Big bluestem) to nutrient addition and herbivore exclusion

Fungal endophytes may alter plant responses to the environment, but how does the environment affect the communities of fungal symbionts within plants? We examined the impact of nutrient addition and herbivore exclusion on endophyte communities of the prairie grass Andropogon gerardii in a full factorial field experiment. Fungi were cultured from stems, young leaves, and mature leaves, ITS sequences obtained, and endophyte incidence, community richness, and composition analyzed. Results indicate that in plots where nutrient addition and herbivore exclusion treatments had been applied separately, fungal endophyte incidence, community composition or evenness did not differ, but that greater species richness was observed in plots with nutrient addition and herbivore exclusion treatments applied in combination, compared to other treatments. Further, although fungal community composition was significantly different in stem and leaf tissues, OTU richness was greater in all endophyte communities in nutrient addition plus herbivore exclusion treatments, regardless of tissue type. Our results indicate the distinct fungal endophyte communities found in different plant tissues respond similarly to environmental factors.     

Patterns of fungal diversity in New Zealand Nothofagus forests

The development of protocols for the conservation of fungi requires knowledge of the factors controlling their distribution, diversity, and community composition. Here we compare patterns of variation in fungal communities across New Zealand's Nothofagus forests, reportedly the most myco-diverse in New Zealand and hence potentially key to effective conservation of fungi in New Zealand. Diversity of leaf endophytic fungi, as assessed by culturing on agar plates, is assessed for three Nothofagus sp. growing in mixed stands from four sites. Host species was found to have a greater influence on fungal community assemblage than site. The leaf endophyte communities associated with Nothofagus solandri and Nothofagus fusca (both Nothofagus subgenus Fuscopora), were more similar to each other than either were to the community associated with Nothofagus menziesii (Nothofagus subgenus Lophozonia). The broad taxonomic groups isolated, identified on the basis of internal transcribed spacer (ITS) sequences, were similar to those found in similar studies from other parts of the world, and from an earlier study on the endophyte diversity in four podocarp species from New Zealand, but there were few matches at species level. Average levels of endophyte species diversity associated with single Nothofagus species and single podocarp species were similar, despite historical literature and collection data recording more than twice as many fungal species on average from the Nothofagus species. The significance of these findings to fungal conservation is discussed.     

Intrastrain internal transcribed spacer heterogeneity in Ganoderma species

Intrastrain internal transcribed spacer (ITS) heterogeneity is first reported from Ganoderma, a fungal genus within Basidiomycetes. ITS amplification products from 4 strains, representing 4 Ganoderma species, were cloned and sequenced. Two to five different ITS types were found within a single strain. The clone sequences were analyzed along with other sequences from Ganoderma retrieved from GenBank. The results show that sequence variation within strains varies considerably with species and the heterogeneity may occur in the 3 parts (ITS1, ITS2, and 5.8S) of the ITS region.     

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.     

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.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

Fungal endophyte communities differ between chestnut galls and surrounding foliar tissues

Foliar endophytic fungi are present in almost all vascular plants. The composition of endophyte communities varies among plant individuals. Likely, but understudied, sources of this variation are the species composition of the plant community and initial attacks by insect herbivores. We addressed these issues by characterizing fungal endophyte communities on leaves of chestnut (Castanea sativa) grown in pure vs. mixed stands. We used ITS metabarcoding methods to identify endophytic fungi associated with galls caused by the invasive gall wasp, Dryocosmus kuriphilus, and with surrounding chestnut leaf tissues. We found 1378 different OTUs. The richness, diversity and composition of endophyte communities differed between galls and surrounding leaf tissues but were independent of forest stand composition. Fungal endophyte richness was lower in galls than in surrounding leaf tissues. Most differences in the composition of fungal endophyte communities between galls and foliar tissues were due to OTU turnover. These results suggest that insect-induced galls provide a particular habitat condition for endophytic microorganisms, regardless of forest species composition. A better understanding of endophyte biology is important to improve their use as biocontrol agents of galling insects.     

三种杓兰根相关真菌多样性和生态功能

【背景】除了菌根真菌(Orchid mycorrhizal fungi,OrMF)外,兰科植物根中还有其它内生真菌,称为根相关真菌(Root-associated fungi,RAF)。【目的】采用分离培养的方法获得同一栖息地针叶林和灌木林两种不同生境西藏杓兰、黄花杓兰和无苞杓兰的RAF菌株,研究其真菌谱系、多样性和生态功能结构。【方法】从杓兰根碎屑中分离RAF,通过总DNA提取、PCR扩增及测序得到ITS(Internaltranscribedspacer)序列;进行系统发育和多样性分析,并通过NCBI数据库比对得到相似性最高序列的注释信息来分析RAF生态学特性。【结果】共分离得到278株RAF,25种OTU类型,包括23个子囊菌门OTU,2个毛霉菌门OTU。RAF物种丰富度分析发现西藏杓兰的较黄花杓兰高,不同生境没有显著差异;不同杓兰物种较不同生境的RAF群落分化程度高。生态功能分析显示25个OTU包括共生型、腐生型和致病型3种营养型,以及外生菌根菌群、植物病原菌群、内生真菌群、动物病原菌群、真菌寄生菌群、杜鹃花类菌根群、未定义的腐生菌群和不确定型8种共位群。【结论】阐明不同生境采集的不同杓兰中RAF的分布特点和生态功能,为未来研究RAF与杓兰属植物的共生关系奠定基础。     

Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages

Although associated with all plants, fungal endophytes (microfungi that live within healthy plant tissues) represent an unknown proportion of fungal diversity. While there is a growing appreciation of their ecological importance and human uses, little is known about their host specificity, geographic structure, or phylogenetic relationships. We surveyed endophytic Ascomycota from healthy photosynthetic tissues of three plant species (Huperzia selago, Picea mariana, and Dryas integrifolia, representing lycophytes, conifers, and angiosperms, respectively) in northern and southern boreal forest (Québec, Canada) and arctic tundra (Nunavut, Canada). Endophytes were recovered from all plant species surveyed, and were present in <1-41% of 2 mm2 tissue segments examined per host species. Sequence data from the nuclear ribosomal internal transcribed spacer region (ITS) were obtained for 280 of 558 isolates. Species-accumulation curves based on ITS genotypes remained non-asymptotic, and bootstrap analyses indicated that a large number of genotypes remain to be found. The majority of genotypes were recovered from only a single host species, and only 6% of genotypes were shared between boreal and arctic communities. Two independent Bayesian analyses and a neighbor-joining bootstrapping analysis of combined data from the nuclear large and small ribosomal subunits (LSUrDNA, SSUrDNA; 2.4 kb) showed that boreal and arctic endophytes represent Dothideomycetes, Sordariomycetes, Chaetothyriomycetidae, Leotiomycetes, and Pezizomycetes. Many well-supported phylotypes contained only endophytes despite exhaustive sampling of available sequences of Ascomycota. Together, these data demonstrate greater than expected diversity of endophytes at high-latitude sites and provide a framework for assessing the evolution of these poorly known but ubiquitous symbionts of living plants.     

Intrasporal variability of ribosomal sequences in the endomycorrhizal fungus Gigaspora margarita

The sequence variability of the ribosomal internal transcribed spacer (ITS) region, which comprises the 5.8 gene and the flanking regions ITS1 and ITS2, was investigated in the arbuscular mycorrhizal fungus Gigaspora margarita. DNA analysis of a multispore preparation and three single spores led to the identification of 11 slightly different sequences (three variants within a single spore), indicating substantial intersporal and intrasporal genetic variability (up to 9% sequence divergence). The sequence variations inside a single spore may be higher than that observed between spores. Even so, primers designed on the ITS1 and ITS2 regions identified Gi. margarita isolates and detected the endophyte during colonization.     

Moths that Vector a Plant Pathogen also Transport Endophytic Fungi and Mycoparasitic Antagonists

Claviceps paspali, a common fungal pathogen of Paspalum grasses, attracts moth vectors by producing sugary exudates in the grass florets it infects. These exudates also support mycoparasitic Fusarium species that may negatively influence C. paspali fitness. We examined the potential for moths on which C. paspali depends to also transmit mycoparasitic Fusarium and fungal endophytes, which inhabit asymptomatic plant tissue and may influence host susceptibility to pathogens. We quantified infections by C. paspali, Fusarium spp., and endophytic fungi associated with Paspalum spp. at focal sites in the southeastern USA and used data from the nuclear internal transcribed spacer (ITS rDNA) to compare communities of plant-associated and moth-borne fungi. ITS sequences of moth-borne fungi were identical to reference sequences of mycoparasitic Fusarium heterosporum and to three distinct endophytic fungi isolated from Paspalum species. Our results demonstrate an unexpected overlap of fungal communities between disparate locations and among plant species and plant tissues, and suggest an unexpected role of moths, which vector a plant pathogen, to transmit other guilds of fungi. In turn, the potential for insects to transmit plant pathogens as well as mycoparasites and endophytic fungi suggests complex interactions underlying a commonly observed grass–pathogen system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.     

Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.     

Fungal Endophytes of Alpinia officinarum Rhizomes: Insights on Diversity and Variation across Growth Years,Growth Sites,and the Inner Active Chemical Concentration

In the present study, the terminal-restriction fragment length polymorphism (T-RFLP) technique, combined with the use of a clone library, was applied to assess the baseline diversity of fungal endophyte communities associated with rhizomes of Alpinia officinarum Hance, a medicinal plant with a long history of use. A total of 46 distinct T-RFLP fragment peaks were detected using HhaI or MspI mono-digestion-targeted, amplified fungal rDNA ITS sequences from A. officinarum rhizomes. Cloning and sequencing of representative sequences resulted in the detection of members of 10 fungal genera: Pestalotiopsis, Sebacina, Penicillium, Marasmius, Fusarium, Exserohilum, Mycoleptodiscus, Colletotrichum, Meyerozyma, and Scopulariopsis. The T-RFLP profiles revealed an influence of growth year of the host plant on fungal endophyte communities in rhizomes of this plant species; whereas, the geographic location where A. officinarum was grown contributed to only limited variation in the fungal endophyte communities of the host tissue. Furthermore, non-metric multidimensional scaling (NMDS) analysis across all of the rhizome samples showed that the fungal endophyte community assemblages in the rhizome samples could be grouped according to the presence of two types of active indicator chemicals: total volatile oils and galangin. Our present results, for the first time, address a diverse fungal endophyte community is able to internally colonize the rhizome tissue of A. officinarum. The diversity of the fungal endophytes found in the A. officinarum rhizome appeared to be closely correlated with the accumulation of active chemicals in the host plant tissue. The present study also provides the first systematic overview of the fungal endophyte communities in plant rhizome tissue using a culture-independent method.     

Molecular analysis of phylogenetic relationships among Myrmecophytic macaranga species (Euphorbiaceae)

Many species of the paleotropical pioneer tree genus Macaranga Thou. (Euphorbiaceae) live in association with ants. Various types of mutualistic interactions exist, ranging from the attraction of unspecific ant visitors to obligate myrmecophytism. In the latter, nesting space and food bodies are exchanged for protection by highly specific ant partners (mainly species of the myrmicine genus Crematogaster). As a first step toward elucidating the coevolution of ant-plant interactions in the Macaranga-Crematogaster system, we have initiated a molecular investigation of the plant partners' phylogeny. Nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were analyzed for 73 accessions from 47 Macaranga species, representing 17 sections or informally described species groups. Three accessions from the putative sister taxon Mallotus Lour, were included as outgroups. Cladograms of the ITS data revealed Macaranga to be nested within Mallotus. ITS sequences are highly similar within section Pachystemon s.str., suggesting a relatively recent and rapid radiation of obligate myrmecophytes within this section. Forty-three accessions, mainly of ant-inhabited species, were additionally investigated by random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR) techniques. Phenetic analysis of RAPD and MP-PCR banding profiles generally confirmed the ITS results. Best resolutions for individual clades were obtained when ITS and RAPD/MP-PCR data were combined into a single matrix and analyzed phenetically. The combined analysis suggests multiple (four) rather than a single evolutionary origin of myrmecophytism, at least one reversal from obligate myrmecophytism to nonmyrmecophytism, and one loss of mutualistic specifity.     

Fungal community analysis by large-scale sequencing of environmental samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized. All sequences were deposited in GenBank, with accession numbers AY 969316 to AY 970290 for the ITS sequences and AY 969135 to AY 969315 for the SSU sequences.     

Relationships between endophyte diversity and leaf optical properties

A single tropical plant species can harbour hundreds of endophyte species within its tissues. Beyond this, little is known about the relationship between endophyte colonization, leaf traits and spectral properties of leaves. We explore these relationships in Coccoloba cereifera, a plant well known for its symbiotic properties. Endophyte richness in C. cereifera was statistically correlated with leaf traits such as water content, the ratio of fresh weight/dry weight and polyphenol/leaf specific weight. Endophyte diversity was also related to spectral vegetation indices of chlorophyll content. The associations among endophyte diversity, leaf traits and spectral reflectance pose new questions and present new opportunities to better understand plant–fungal symbioses and related leaf optical properties.     

An endophyte‐rich diet increases ant predation on a specialist herbivorous insect

1. All plants form symbioses with microfungi, known as endophytes, which live within plant tissues. Numerous studies have documented endophyte–herbivore antagonism in grass systems, but plant–endophyte–insect interactions are highly variable for forbs and woody plants. 2. The net effect of endophytes on insect herbivory may be modified by their interactions with higher trophic levels, such as predators. Including these multitrophic dynamics may explain some of the variability among endophyte studies of non‐grass plants, which are currently based exclusively on bitrophic studies. 3. The abundance of natural foliar endophytes in a Neotropical vine was manipulated and beetles were fed high or low endophyte diets. Experimental assays assessed whether dietary endophyte load affected beetle growth, leaf consumption, and susceptibility to ant predation. 4. Beetles feeding on high‐ versus low‐endophyte plants had almost identical growth and leaf consumption rates. 5. In a field bioassay, however, it was discovered that feeding on an endophyte‐rich diet increased a beetle's odds of capture by predatory ants nine‐fold. 6. Endophytes could thus provide an indirect, enemy‐mediated form of plant defence that operates even against specialist herbivores. We argue that a multitrophic approach is necessary to untangle the potentially diverse types of endophyte defence among plants.