Foliar Endophytes Research Articles

Detection of foliar endophytes and their metabolites in Picea and Pinus seedling needles

Abstract The needles of Picea glauca (white spruce) and Pinus strobus (white pine) trees infected with toxigenic fungal endophytes contain varying concentrations of their secondary metabolites that are toxic to either insect pests or needle pathogens. In the present study, liquid chromatography-mass spectrometric methods to determine needle concentrations of metabolites of four endophyte species were developed. The endophytes considered were a Phialocephala sp. (vermiculine) and Phialocephala scopiformis (rugulosin) from white spruce, as well as a Xylaria sp. (griseofulvin) and Lophodermium nitens (pyrenophorol) from white pine needles. To ensure that needles were infected with the associated fungal endophyte, suitable qPCR-based methods were also developed. There was a high degree of concordance between the qPCR analysis of the fungal mycelium and the LC-MS/MS quantification of the associated metabolites. Concentrations of the antifungal compounds griseofulvin and pyrenophorol were present in amounts that affect conifer needle diseases including white pine blister rust caused by Cronartium ribicola. Similarly, concentrations of the antiinsectan compounds vermiculine and rugulosin were in the range known to reduce the growth of Choristoneura fumiferana and mitigate foliage damage.

Sydowia polyspora is both a Foliar Endophyte and a Preemergent Seed Pathogen in Pinus ponderosa.

Poor seedling performance and reduced seed emergence are often ascribed to known pathogens that cause low seedling recruitment and poor seed emergence in forest nurseries and regeneration plantings. On the other hand, foliar endophytes are often overlooked as a source of poor emergence or tree seedling disease. Here, we show that an endophytic fungus common to the foliar microbiome of Pinus ponderosa acts as a cryptic pathogen in delaying emergence. In a series of experiments, we inoculated seed of P. ponderosa with a suspension of Sydowia polyspora 12 h prior to sowing. S. polyspora reduced seed emergence of its host, P. ponderosa, by as much as 30%. A tetrazolium chloride viability assay showed that S. polyspora reduces emergence by preventing germination; seed remained viable. In sum, pathogens affecting tree seed emergence and seedling recruitment may be endophytic as well as in seed and soil and deserve greater attention in studies of natural regeneration.

Biogeography of plant‐associated fungal symbionts in mountain ecosystems: A meta‐analysis

AbstractAimPredicting the potential for climate change to disrupt host–microbe symbioses requires basic knowledge of the biogeography of these consortia. In plants, fungal symbionts can ameliorate the abiotic stressors that accompany climate warming and thus could influence plants under a changing climate. Forecasting future plant–microbe interactions first requires knowledge of current fungal symbiont distributions, which are poorly resolved relative to the distributions of plants.LocationWe used meta‐analysis to summarize the biogeographic distributions of plant‐fungal symbionts in mountain ecosystems worldwide, because these ecosystems are likely to be among the first to experience climate change‐induced range shifts.MethodsWe analysed 374 records from 53 publications to identify general trends, pinpoint areas in need of greater study and develop reporting guidelines to facilitate future syntheses.ResultsElevational patterns varied strongly among fungal and plant functional groups. Fungal diversity and abundance increased with altitude for the ectomycorrhizal fungi. However, arbuscular mycorrhizal fungi and localized foliar endophytes declined in either abundance or diversity with altitude. In shrubs, fungal abundance increased with elevation, but in C3 grasses, fungal abundance declined with elevation. Altitudinal patterns in fungal composition were stronger than gradients in fungal abundance or diversity, suggesting that species turnover contributes more to elevational gradients in fungal symbionts than does variation in abundance or richness. Plant functional groups were overrepresented by C3 grasses and trees, with surprisingly few data on sedges or shrubs, despite their ecological dominance in mountain ecosystems. Similarly, epichloae, ericoid mycorrhizal fungi and root endophytes were understudied relative to other fungal groups.Main ConclusionsMeta‐analysis revealed broad biogeographic patterns in plant‐fungal symbiont abundance, diversity and composition that inform predictions of future distributions.

Does a foliar endophyte improve plant fitness under flooding?

Although endophytic fungi are ubiquitous in plants, their full range of ecological effects has yet to be characterized, particularly in non-agronomic systems. In this study, we compared the responses of two congeneric bluegrass species to flooding. Both plant species co-occur in subalpine zones of the Rocky Mountains. Marsh bluegrass (Poa leptocoma) commonly hosts a vertically transmitted fungal endophyte (Epichloe sp.) and naturally grows in wetter conditions than does nodding bluegrass (Poa reflexa), which lacks an epichloid endophyte. We investigated the novel hypothesis that endophyte symbiosis promotes host fitness under flooded conditions, contributing to niche differentiation between the two bluegrass species. We used a factorial greenhouse experiment to test whether endophyte presence improved survival, growth, or reproduction of P. leptocoma under flooded versus non-flooded edaphic conditions by experimentally removing the endophyte from half of the plants. We compared P. leptocoma responses to those of the endophyte-free congener. In contrast to expectations generated from the natural distributions of the two plant species, endophyte presence was more beneficial to P. leptocoma under ambient soil moisture than under flooding. Increased benefits of symbiosis in drier soils are consistent with studies of other grass endophytes. Flooded soils also unexpectedly improved the growth of P. reflexa more than that of the wet habitat specialist, P. leptocoma. While our results demonstrate an overall benefit of fungal symbiosis in this system, ecological factors other than flooding per se likely underlie the observed geographical distributions of these congeneric grasses in nature.

Absence of genome reduction in diverse, facultative endohyphal bacteria.

Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.

Isolation of peat swamp forest foliar endophyte fungi as biofertilizer

<p><span>Peatland restoration activity is facing many obstacles, particularly in planting techniques and poor nutrient in peat soil. Naturally, endophytic fungi are abundant and have great potential as biofertilizer. This research investigates the potential endophytic fungi isolated from leaves of peat swamp tree species for biofertilizer. Research activities include: exploration, in vitro test to examine the phosphate solubilization and identification. Result showed that there were 360 leave segments collected from 4 sampling locations. The colonization percentage of 222 isolates ranged from 52.17% - 60.17%. Fifty seven morphospecies were selected from 222 isolates. Twelve isolates demonstrated ability to produce clear zones and ten isolates were selected for identification. It is concluded that twelve isolated demonstrated potential ability to produce clear zone and <em>Penicillum citrinum</em> isolate P3.10 was identified as an isolate that show the highest potential ability as a biofertilizer</span></p>

Pestalotiopsis species occur as generalist endophytes in trees of Western Ghats forests of southern India

One hundred tree species from 4 different forest types (25 from each forest) of the Western Ghats were studied for the occurrence of Pestalotiopsis as foliar endophytes. Morphological and ITS sequence study confirmed that species of Pestalotiopsis are generalist endophytes infecting taxonomically unrelated tree hosts. Furthermore, a single tree species harboured more than one species of this endophyte. Considering the leaf as a microhabitat, such a wide ecological amplitude of Pestalotiopsis as a foliar endophyte appears to be the result of accumulation of a suite of traits which are governed by the environment. This could be one of the reasons for the existence of generalist fungi among endophytes transcending host taxonomic and habitat restrictions.

Three symbionts involved in interspecific plant-soil feedback: epichloid endophytes and mycorrhizal fungi affect the performance of rhizobia-legume symbiosis

Aims Plants interact by modifying soil conditions in plant-soil feedback processes. Foliar endophytes of grasses exert multiple effects on host rhizosphere with potential consequences on plant-soil feedback. Here, we hypothesize that the grass-endophyte symbiosis impairs soil symbiotic potential, and in turn influences legume performance and nitrogen acquisition.

Data on litter quality of host grass plants with and without fungal endophytes.

Certain Pooideae species form persistent symbiosis with fungal endophytes of Epichloë genus. Although endophytes are known to impact the ecology and evolution of host species, their effects on parameters related with quality of plant biomass has been elusive. This article provides information about parameters related with the quality of plant litter biomass of two important grass species (Schedonorus phoenix and Schedonorus pratensis) affected by the symbiosis with fungal endophytes (Epichloë coenophiala and Epichloë uncinata, respectively). Four population origins of S. phoenix and one of S. pratensis were included. Mineral, biochemical and structural parameters were obtained from three samples per factors combination [species (and population origin)×endophyte]. This data can be potentially used in other studies which, by means of ‘data reanalyzing’ or meta-analysis, attempt to find generalizations about endophyte effects on host plant litter biomass. The present data is associated with the research article “Role of foliar fungal endophytes on litter decomposition among species and population origins” (Gundel et al., In preparation) [1].

Sexual and asexual states of some endophytic Phialocephala species of Picea

Unidentified DNA sequences in isolation-based or culture-free studies of conifer endophytes are a persistent problem that requires a field approach to resolve. An investigation of foliar endophytes of Picea glauca, P. mariana, P. rubens and Pinus strobus in eastern Canada, using a combined field, morphological, cultural and DNA sequencing approach, resulted in the frequent isolation of Phialocephala spp. and the first verified discovery of their mollisia-like sexual states in the field. Phialocephala scopiformis and Ph. piceae were the most frequent species isolated as endophytes from healthy conifer needles. Corresponding Mollisia or mollisioid sexual states for Ph. scopiformis, Ph. piceae and several undescribed species in a clade containing Ph. dimorphospora were collected in the sampling area and characterized by analysis of the nuc internal transcribed spacer rDNA (ITS) and gene for the largest subunit of RNA polymerase II (RPB1) loci. Four novel species and one new combination in a clade containing Ph. dimorphospora, the type of Phialocephala, are presented, accompanied by descriptions of apothecia and previously undocumented synanamorphs. An epitype culture and corresponding reference sequences for Phialocephala dimorphospora are proposed. The resulting ITS barcodes linked with robust taxonomic species concepts are an important resource for future research on forest ecosystems and endophytes.

Full Genome of Phialocephala scopiformis DAOMC 229536, a Fungal Endophyte of Spruce Producing the Potent Anti-Insectan Compound Rugulosin.

We present the full genome of Phialocephala scopiformis DAOMC 229536 (Helotiales, Ascomycota), a foliar endophyte of white spruce from eastern Quebec. DAOMC 229536 produces the anti-insectan compound rugulosin, which inhibits a devastating forestry pest, the spruce budworm. This genome will enable fungal genotyping and host-endophyte evolutionary genomics in inoculated trees.

Positive plant–soil feedbacks of the invasive Impatiens glandulifera and their effects on above‐ground microbial communities

SummaryImpatiens glandulifera is one of the most widespread invasive plant species in the UK. Although aspects of its biology are known, there is little information about its association with microbial communities, both above ground and below ground. Furthermore, it is unknown whether this species exhibits any form of plant–soil feedback (PSF), commonly seen in other invasive weeds. We conducted a PSF experiment, in which plants of I. glandulifera were grown in soil that supported the species and compared with plants grown in a control soil from the same locality. Soil nutrients were measured, and the soil and foliar microbial communities were assessed. Impatiens glandulifera grew larger and faster in conditioned soil compared with the control. Higher levels of phosphate were also found in conditioned soils. Arbuscular mycorrhizal fungal (AMF) colonisation was lower in conditioned soils, suggesting that I. glandulifera may rapidly alter AMF communities in invaded areas. PSFs had a significant effect on the foliar endophyte community, with clear separation of species between conditioned and control soils. These results show that I. glandulifera displayed a positive PSF and the PSF mechanism extended beyond the soil microbial community to affect foliar endophytes. The observed increase in endophytes in plants grown in conditioned soil could enhance resistance to herbivory, thus further accentuating the invasive properties of this species.

Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer.

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N2 -fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P.flexilis-AAB association is consistent across years, we re-sampled P.flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N2 , we incubated twigs with (13) N2 -enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using (13) N. We used the acetylene reduction assay to test for nitrogenase activity within P.flexilis twigs four times from June to September. We found evidence for N2 fixation in P.flexilis foliage. N2 diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8-13.6μgNm(-2) d(-1) to P.flexilis stands. AAB dominated the P.flexilis needle endophyte community. We propose that foliar endophytes represent a low-cost, evolutionarily stableN2 -fixing strategy for long-lived conifers. This novel source of biological N2 fixation has fundamental implications for understanding forest N budgets.

Spatial Ecology of the Fungal Genus Xylaria in a Tropical Cloud Forest

AbstractFungal symbioses with plants are ubiquitous, ancient, and vital to both ecosystem function and plant health. However, benefits to fungal symbionts are not well explored, especially in non‐mycorrhizal fungi. The Foraging Ascomycete hypothesis proposes that some wood‐decomposing fungi may shift life‐history strategies to endophytism to bridge gaps in time and space between suitable substrates. To test this hypothesis we examine spatial relationships of Xylaria endophytic fungi in the forest canopy with Xylaria decomposer fungi on the forest floor. We sampled for fungi of the genus Xylaria using a spatially explicit sampling scheme in a remote Ecuadorian cloud forest, and concurrently carried out an extensive culture‐based sampling of fungal foliar endophytes. We found 36 species of Xylaria in our 0.5 ha plot, 31 of which were found to only occur as fruiting bodies. All five species of Xylaria found as endophytes were also found as fruiting bodies. We also tested the relationships of both stages of these fungi to environmental variables. Decomposer fungi were differentiated by species‐specific habitat preferences, with three species being found closer to water than expected by chance. In contrast, endophytes displayed no sensitivity to environmental conditions, such as host, moisture, or canopy cover. We found evidence of spatial linkage between life stages in two species. We also demonstrate that direct transmission of endophytes from leaves to woody substrates is possible. These results indicate that endophytism may represent one way for decomposer fungi to escape moisture limitation, and that endophytic fungi may act as sources of dispersal for decomposer fungi consistent with predictions of the Foraging Ascomycete hypothesis.

The herbaceous landlord: integrating the effects of symbiont consortia within a single host.

Plants are typically infected by a consortium of internal fungal associates, including endophytes in their leaves, as well as arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) in their roots. It is logical that these organisms will interact with each other and the abiotic environment in addition to their host, but there has been little work to date examining the interactions of multiple symbionts within single plant hosts, or how the relationships among symbionts and their host change across environmental conditions. We examined the grass Agrostis capillaris in the context of a climate manipulation experiment in prairies in the Pacific Northwest, USA. Each plant was tested for presence of foliar endophytes in the genus Epichloë, and we measured percent root length colonized (PRLC) by AMF and DSE. We hypothesized that the symbionts in our system would be in competition for host resources, that the outcome of that competition could be driven by the benefit to the host, and that the host plants would be able to allocate carbon to the symbionts in such a way as to maximize fitness benefit within a particular environmental context. We found a correlation between DSE and AMF PRLC across climatic conditions; we also found a fitness cost to increasing DSE colonization, which was negated by presence of Epichloë endophytes. These results suggest that selective pressure on the host is likely to favor host/symbiont relationships that structure the community of symbionts in the most beneficial way possible for the host, not necessarily favoring the individual symbiont that is most beneficial to the host in isolation. These results highlight the need for a more integrative, systems approach to the study of host/symbiont consortia.

Bacterial endophyte communities in the foliage of coast redwood and giant sequoia.

The endophytic bacterial microbiome, with an emerging role in plant nutrient acquisition and stress tolerance, is much less studied in natural plant populations than in agricultural crops. In a previous study, we found consistent associations between trees in the pine family and acetic acid bacteria (AAB) occurring at high relative abundance inside their needles. Our objective here was to determine if that pattern may be general to conifers, or alternatively, is more likely restricted to pines or conifers growing in nutrient limited and exposed environments. We used 16S rRNA pyrosequencing to characterize the foliar endophyte communities of two conifers in the Cupressaceae family: Two coast redwood (CR; Sequoia sempervirens) populations and one giant sequoia (GS; Sequoiadendron giganteum) population were sampled. Similar to the pines, the endophyte communities of the giant trees were dominated by Proteobacteria, Firmicutes, Acidobacteria, and Actinobacteria. However, although some major operational taxonomic units (OTUs) occurred at a high relative abundance of 10–40% in multiple samples, no specific group of bacteria dominated the endophyte community to the extent previously observed in high-elevation pines. Several of the dominating bacterial groups in the CR and GS foliage (e.g., Bacillus, Burkholderia, Actinomycetes) are known for disease- and pest suppression, raising the possibility that the endophytic microbiome protects the giant trees against biotic stress. Many of the most common and abundant OTUs in our dataset were most similar to 16S rRNA sequences from bacteria found in lichens or arctic plants. For example, an OTU belonging to the uncultured Rhizobiales LAR1 lineage, which is commonly associated with lichens, was observed at high relative abundance in many of the CR samples. The taxa shared between the giant trees, arctic plants, and lichens may be part of a broadly defined endophyte microbiome common to temperate, boreal, and tundra ecosystems.

Pervasive Effects of Wildfire on Foliar Endophyte Communities in Montane Forest Trees.

Plants in all terrestrial ecosystems form symbioses with endophytic fungi that inhabit their healthy tissues. How these foliar endophytes respond to wildfires has not been studied previously, but is important given the increasing frequency and intensity of severe wildfires in many ecosystems, and because endophytes can influence plant growth and responses to stress. The goal of this study was to examine effects of severe wildfires on endophyte communities in forest trees, with a focus on traditionally fire-dominated, montane ecosystems in the southwestern USA. We evaluated the abundance, diversity, and composition of endophytes in foliage of Juniperus deppeana (Cupressaceae) and Quercus spp. (Fagaceae) collected contemporaneously from areas affected by recent wildfire and paired areas not affected by recent fire. Study sites spanned four mountain ranges in central and southern Arizona. Our results revealed significant effects of fires on endophyte communities, including decreases in isolation frequency, increases in diversity, and shifts in community structure and taxonomic composition among endophytes of trees affected by recent fires. Responses to fire were similar in endophytes of each host in these fire-dominated ecosystems and reflect regional fire-return intervals, with endophytes after fire representing subsets of the regional mycoflora. Together, these findings contribute to an emerging perspective on the responses of diverse communities to severe fire, and highlight the importance of considering fire history when estimating endophyte diversity and community structure for focal biomes.

Using direct amplification and next-generation sequencing technology to explore foliar endophyte communities in experimentally inoculated western white pines

Abstract Fungal endophytes can influence survivability and disease severity of trees. Here we characterized the endophyte community in Pinus monticola (western white pine), an important species in the northwest USA, largely decimated by pathogenic fungi. We also assessed the ability to successfully inoculate seedlings with desirable endophytes, with the long-term goal of providing a protective microbiome and added defense from pathogens. P. monticola seedlings were inoculated in the field with potential pathogen antagonists and fungi isolated from healthy mature trees. Following inoculations direct amplification and next generation sequencing were used to characterize fungal endophyte communities, and explore interspecific competition, diversity, and co-occurrence patterns in needle tissues. Negative co-occurrence patterns between inoculated fungi and potential pathogens, as well as many other species, suggest early competitive interactions. Our study explores early endophyte community assemblage and shows that fungal inoculations may influence tree growth.

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.

Biotic and abiotic predictors of fungal colonization in grasses of the Colorado Rockies

AbstractAimFungal symbionts are ubiquitous in plants and can mitigate abiotic stressors associated with climate change. Predicting fungal symbiont distributions under future climates first requires knowledge of current distributions and their potential drivers.LocationWe documented colonization by fungal symbionts in perennial, cool‐season grasses along altitudinal gradients in the Rocky Mountains of Colorado, USA.MethodsAcross seven replicate altitudinal gradients, spanning c. 1400 vertical meters, we scored fungal colonization for 46 grass species. We documented altitudinal clines in colonization by both above‐ground and below‐ground fungal symbionts for the first time, including localized foliar endophytes (LFE) and systemic endophytes (epichloae) in leaves and arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) in roots. For a subset of 16 well‐sampled grass species, we used model selection procedures to evaluate the relative importance of geography, edaphic factors and host plant identity. We also assessed the influence of host phylogenetic relatedness and colonization by co‐infecting fungi.ResultsLevels of fungal colonization varied strongly with host plant identity, but the effects of particular host species were not consistent across fungal groups. In addition to the influence of host identity, epichloae colonization declined with elevation and varied with geography (latitude/longitude) and edaphic factors. Geography and collection date were important predictors of LFE colonization, with higher colonization later in the growing season. Colonization estimates for the obligately plant‐associated fungi (epichloae, AMF) were phylogenetically conserved across the grass supertree. Positive correlations between AMF and DSE, which remained even after accounting for host plant relatedness, suggested possible synergisms between these fungal groups.Main conclusionsOur survey showed greater host specificity in patterns of fungal colonization than prior reports and revealed that different fungal symbiont groups do not share similar drivers. Conserving plant–fungal symbioses under future climates may require unique strategies for different plant species and fungal symbiont types.