Spatio-temporal dynamics of endophyte diversity in the canopy of European ash (Fraxinus excelsior)

Leaf-inhabiting endophytic fungi of Fraxinus excelsior growing in a floodplain forest were isolated during 2008 to investigate vertical community structure, species richness and seasonal variation. The analysis of 848 fungal endophytes from 213 leaves resulted in 50 different species. In the understorey, infection density and species richness were higher than in the crowns of mature trees throughout the whole vegetation period. Within tree crowns, sun-exposed leaves of the top canopy exhibited the lowest infection rates. Most species were rare or absent in spring and in the light crowns and frequent in autumn and the understorey. However, some species, especially the two most frequent, Alternaria infectoria and A. alternata, deviated from these patterns. Young leaves were nearly free of endophytes. Apparently, the subsequent infection and establishment of fungi strongly depend on microclimatic parameters and leaf characters, which create highly variable spatial and temporal colonisation patterns within an individual tree.     

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.     

Diversity and dynamics of fungal endophytes in leaves, stems and roots of Stellera chamaejasme L. in northwestern China

This study was conducted to explore fungal endophyte communities inhabiting a toxic weed (Stellera chamaejasme L.) from meadows of northwestern China. The effects of plant tissue and growth stage on endophyte assemblages were characterized. Endophytes were recovered from 50 % of the samples, with a total of 714 isolates. 41 operational taxonomical units (OTUs) were identified, consisting of 40 OTUs belonging primarily to Ascomycota and 1 OTU belonging to Basidiomycota. Pleosporales and Hypocreales were the orders contributing the most species to the endophytic assemblages. The total colonization frequency and species richness of endophytic fungi were higher in roots than in leaves and stems. In addition, for the plant tissues, the structure of fungal communities differed significantly by growth stages of leaf emergence and dormancy; for the plant growth stages, the structure of fungal communities differed significantly by plant tissues. This study demonstrates that S. chamaejasme serves as a reservoir for a wide variety of fungal endophytes that can be isolated from various plant tissues.     

Fungal endophyte communities in above- and belowground olive tree organs and the effect of season and geographic location on their structures

Studies comparing fungal endophytes between above- and belowground woody crop organs and the factors that may structure their communities are lacking. Due to its great impact on the Mediterranean Basin, the olive tree was chosen for the isolation of endophytic fungi from roots, leaves and twigs in two seasons in north-eastern Portugal. Nine hundred seventy-six isolates belonging to 38 fungal species were obtained. Phomopsis columnaris, Fusarium oxysporum and Trichoderma gamsii were the most frequently isolated, collectively representing 69% of the isolates. Fungal diversity in the roots was higher than in the aboveground organs and higher in spring than autumn. Endophyte community similarity between the above- and belowground organs and between seasons was low. Species composition also varied spatially, with the fungal composition of the roots varying more among locations than that of the aboveground organs. Our results suggest olive tree endophyte community structure is affected by plant organ, location and season.     

Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity

Calotropis procera has many important medicinal properties with proven pharmacological potential. Some of these properties may be mediated by its fungal endophytes. This study analyzed, for the first time, the community of endophytic fungi of C. procera outside its region of origin. A total of 156 fungal isolates distributed across 19 taxa were obtained from 468 fragments of C. procera leaves at different stages of maturation. The rate of endophyte colonization increased with the leaf age/development. The dominant species of endophytic fungi of C. procera introduced in Northeast Brazil were different from those found in studies on the same species and other species of the same genus in native regions. The dominant endophyte was Phaeoramularia calotropidis (63.5 %), followed by Guignardia bidwellii (21.1 %). Six isolates of endophytic fungi showed antimicrobial activity against human pathogenic micro-organisms and one plant pathogenic fungus. The antibacterial activity was more intense than the antifungal activity. The endophytic Curvularia pallescens (URM 6048) stood out inhibited Gram-positive bacteria, Staphylococcus aureus, Streptococcus pyogenes, the plant pathogenic fungus Colletotrichum dematium. Ecological and biotechnological aspects of endophytic mycota are discussed.     

Fungal endophytes from cactus Cereus jamacaru in Brazilian tropical dry forest: a first study

Endophytic fungi live within the healthy tissues of plants and can promote host species tolerance to different environmental stresses. However, most studies have been of plants in humid environments, and there are few reports of the benefits of such associations for plants of extreme environments. Our aims were to identify endophytic fungi using morphological taxonomy, to explore richness and estimate species frequency in a cactus, C. jamacaru, in Brazilian tropical dry forest (Caatinga). We thus identified 59 taxa, corresponding to 69.7 % of the total number of isolates; the other 30.3 % were sterile mycelia. Cladosporium cladosporioides and Fusarium oxysporum were the species most commonly isolated, followed by Acremonium implicatum, Aureobasidium pullulans, Trichoderma viride, Chrysonilia sitophila, and Aspergillus flavus. Forty-seven species are recorded for the first time as endophytic fungi of cacti, and 18 others as endophytes for Brazil; this suggests that C. jamacaru harbors a highly diverse fungal community as measured by a diversity index. However, species accumulation curves suggest that our study still underestimates endophyte diversity because it does not provide an exhaustive sample. To our knowledge, this is the first report of endophytic fungi from C. jamacaru in tropical dry forests.     

Endophytic fungi associated with Macrosolen tricolor and its host Camellia oleifera

Endophytic fungi play an important role in terrestrial ecosystem, while little is known about those in hemi-parasitic plants, a group of special plants which absorb nutrients from its hosts by haustoria. The relationship of the endophytes in the two parts of the bipartite systems (hemiparasites together with their hosts) is also poorly understood. Endophytic fungi of a hemi-parasitic plant Macrosolen tricolor, and its host plant Camellia oleifera were investigated and compared in this study. M. tricolor contained rich and diversified endophytic fungi (H′ = 2.829), which consisted mainly of ascomycetes, distributed in more than ten orders of four classes (Sordariomycetes, Dothideomycetes, Leotiomycetes and Eurotiomycetes) besides Incertae sedis strains (23.2 % of total). In addition, 2.2 % of isolates were identified to be Basidiomycota, all of which belonged to Agaricomycetes. Obvious differences were observed between the endophytic fungal assembles in the leaves and those in the branches of M. tricolor. The endophytic fungi isolated from C. oleifera distributed in nearly same orders of the four classes of Ascomycota and one class (Agaricomycetes) of Basidiomycota as those from M. tricolor with similar proportion. For both M. tricolor and C. oleifera, Valsa sp. was the dominant endophyte species in the leaves, Torula sp. 1 and Fusarium sp. 1 were the dominant endophytic fungi in the branches. The similarity coefficient of the endophyte assembles in the two host was 64.4 %. Canonical correspondence analysis showed that the endophyte assembles of M. tricolor and C. oleifera were significantly different (p < 0.01).     

Isolation of endophytes from leaves of <Emphasis Type="Italic">Neolitsea sericea</Emphasis> in broadleaf and conifer stands

Endophytic fungi were isolated from leaves of Neolitsea sericea, a major lauraceous tree in the laurel forests of southern Kyushu, collected from the understory layer of broadleaf and conifer stands. Cytosphaera sp. and a species of Ascomycetes in leaf blade segments, plus a xylariaceous species and Phomopsis spp. in petiole segments, were isolated at relatively high frequency. In general, isolation frequencies of endophytes were higher in petiole than blade segments. In blade segments, patterns of endophyte isolation were quite different among stands, while relatively similar in petiole segments. Significant effects of sampling sites or canopy vegetation were rarely detected. the understory layer of laurel forests. Neolitsea sericea is an evergreen broadleaf lauraceous tree, widely distributed in areas of eastern Asia. In southern Kyushu, it is one of the most common trees, growing both as canopy and understory species. In this study, endophytes were isolated from the leaves of N. sericea growing in the understory layer of conifer and broadleaf forest stands to survey the endophytes of N. sericea leaves and to examine the effect of the canopy layer on endophytic mycobiota in understory plants.     

Two fungal symbioses collide: endophytic fungi are not welcome in leaf-cutting ant gardens

Interactions among the component members of different symbioses are not well studied. For example, leaf-cutting ants maintain an obligate symbiosis with their fungal garden, while the leaf material they provide to their garden is usually filled with endophytic fungi. The ants and their cultivar may interact with hundreds of endophytic fungal species, yet little is known about these interactions. Experimental manipulations showed that (i) ants spend more time cutting leaves from a tropical vine, Merremia umbellata, with high versus low endophyte densities, (ii) ants reduce the amount of endophytic fungi in leaves before planting them in their gardens, (iii) the ants'' fungal cultivar inhibits the growth of most endophytes tested. Moreover, the inhibition by the ants'' cultivar was relatively greater for more rapidly growing endophyte strains that could potentially out-compete or overtake the garden. Our results suggest that endophytes are not welcome in the garden, and that the ants and their cultivar combine ant hygiene behaviour with fungal inhibition to reduce endophyte activity in the nest.     

Geographic and Tissue Influences on Endophytic Fungal Communities of Taxus chinensis var. mairei in China

Endophytc fungi were collected from the barks, branches and leaves of Taxus chinensis var. mairei from the Jiangxi, Zhejiang and Chongqing regions of China and their influences on geographic and tissue investigated. A total of 145 fungal taxa were identified based on molecular techniques, of these 125 taxa (86.2 %) belonging to Ascomycota, 14 (9.7 %) to Basidiomycota, 5 (3.4 %) to Zygomycota, and 1 (0.7 %) to undefined fungi. The species richness and diversity of endophytic fungi were significantly affected by tissue, and were 1.2–2.5-fold higher in the branches and barks when compared to the leaves. The locality affected the species richness per tree and the shannon diversity index per tree by longitude. The endophyte assemblages were strongly shaped by locality and tissue according to partial least squares discriminant analysis. In addition, the distributions of dominant fungi at orders and genera levels differed as a function of locality and tissue. Most of the dominant taxa showed spatial heterogeneity and tissue specificity or preference and many fungal taxa with low frequency were special to one locality or one tissue.     

Vertical transmission of fungal endophytes is widespread in forbs

To date, it has been thought that endophytic fungi in forbs infect the leaves of their hosts most commonly by air‐borne spores (termed “horizontal transmission”). Here, we show that vertical transmission from mother plant to offspring, via seeds, occurs in six forb species (Centaurea cyanus, C. nigra, Papaver rhoeas, Plantago lanceolata, Rumex acetosa, and Senecio vulgaris), suggesting that this may be a widespread phenomenon. Mature seeds were collected from field‐grown plants and endophytes isolated from these, and from subsequent cotyledons and true leaves of seedlings, grown in sterile conditions. Most seeds contain one species of fungus, although the identity of the endophyte differs between plant species. Strong evidence for vertical transmission was found for two endophyte species, Alternaria alternata and Cladosporium sphaerospermum. These fungi were recovered from within seeds, cotyledons, and true leaves, although the plant species they were associated with differed. Vertical transmission appears to be an imperfect process, and germination seems to present a bottleneck for fungal growth. We also found that A. alternata and C. sphaerospermum occur on, and within pollen grains, showing that endophyte transmission can be both within and between plant generations. Fungal growth with the pollen tube is likely to be the way in which endophytes enter the developing seed. The fact that true vertical transmission seems common suggests a more mutualistic association between these fungi and their hosts than has previously been thought, and possession of endophytes by seedling plants could have far‐reaching ecological consequences. Seedlings may have different growth rates and be better protected against herbivores and pathogens, dependent on the fungi that were present in the mother plant. This would represent a novel case of trans‐generational resistance in plants.     

A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district,eastern Japan

To clarify the effects of forest fragmentation and a change in tree species composition following urbanization on endophytic fungal communities, we isolated fungal endophytes from the foliage of nine tree species in suburban (Kashiwa City, Chiba) and rural (Mt. Wagakuni, Ibaraki; Mt. Takao, Tokyo) forests and compared the fungal communities between sites and host tree species. Host specificity was evaluated using the index of host specificity (Si), and the number of isolated species, total isolation frequency, and the diversity index were calculated. From just one to several host-specific species were recognized in all host tree species at all sites. The total isolation frequency of all fungal species on Quercus myrsinaefolia, Quercus serrata, and Chamaecyparis obtusa and the total isolation frequency of host-specific species on Q. myrsinaefolia, Q. serrata, and Eurya japonica were significantly lower in Kashiwa than in the rural forests. The similarity indices (nonmetric multidimensional scaling (NMS) and C_MH) of endophytic communities among different tree species were higher in Kashiwa, as many tree species shared the same fungal species in the suburban forest. Endophytic fungi with a broad host range were grouped into four clusters suggesting their preference for conifer/broadleaves and evergreen/deciduous trees. Forest fragmentation and isolation by urbanization have been shown to cause the decline of host-specific fungal species and a decrease in β diversity of endophytic communities, i.e., endophytic communities associated with tree leaves in suburban forests were found to be depauperate.     

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

There 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.     

Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichloë amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

Effects of simultaneous infections of endophytic fungi and arbuscular mycorrhizal fungi on the growth of their shared host grass Achnatherum sibiricum under varying N and P supply

Grasses can be infected by endophytic fungi and arbuscular mycorrhizal (AM) fungi simultaneously. Here, we investigated the interactions of a native grass, Achnatherum sibiricum, with both endophytic and AM fungi (Glomus mosseae, GM and Glomus etunicatum, GE) at different nitrogen (N) and phosphorus (P) levels. The results showed that endophyte infection significantly suppressed the colonization rates and spore density of GE, but had no effect on those of GM. Endophyte infection increased shoot biomass regardless of the nutrient conditions. The effects of AM fungi on host growth were dependent on mycorrhizal species. There was no significant interaction between endophytic fungi and GE on host growth; however, a significant interaction between endophytic fungi and GE occurred in total phenolic concentrations and P uptake. As for GM, a significant interaction among endophytic fungi, AM fungi and nutrient availability occurred in shoot growth. Under sufficient N and P conditions, endophyte infection alleviated the detrimental effects of GM colonization on host growth.     

Foliar nutrients shape fungal endophyte communities in Western white pine (Pinus monticola) with implications for white-tailed deer herbivory

Asymptomatic fungal endophytes colonize tissues of woody plants worldwide, with largely unknown ecological effects. Using culture-based methods and ITS1-5.8S-ITS2 rDNA sequence analysis, we investigated differences between foliar endophyte communities in disease-resistant hybrid and wild-type Pinus monticola (Western white pine) trees with observed variation in tree growth, vigor, and browsing damage by white-tailed deer (Odocoileus virginianus). We isolated 69 phylotypes of endophytic fungi in at least 39 genera, including 26 that have not previously been reported in P. monticola. Principal components analysis revealed that endophyte communities differed between browsed seedlings, unbrowsed seedlings, and unbrowsed adult trees. Sulfur, nitrate and calcium concentrations correlated with endophyte community differences among tree groups based on a distance-based redundancy analysis. Our results indicate that foliar nutrient variation influences endophyte community assembly and deer herbivory in P. monticola on a small landscape scale (80 hectares).     

Endophytic and epiphytic phyllosphere fungi of Camellia japonica: seasonal and leaf age-dependent variations

Seasonal and leaf age-dependent variations in the endophytic and epiphytic phyllosphere fungal assemblages of Camellia japonica were examined and compared. Live leaves of C. japonica were collected in four seasons (May, Aug, Nov, Feb), and fungi were isolated from healthy-looking leaves of 0, 1, 2 and 3 y old. The infection rate and total number of endophytic fungi increased May-Feb, and species richness of endophytes increased as leaves aged. In contrast the infection rate of epiphytic fungi was 100% for all leaf ages at every sampling date. The total number of epiphytic fungi isolated was greatest in May and lowest in Aug. The species richness of epiphytes did not differ significantly by season or leaf age. Eight fungal species were recorded as major phyllosphere fungi of C. japonica. Seasonal variations were detected for the frequencies of Colletotrichum gloeosporioides, C. acutatum, and epiphytes Pestalotiopsis sp.1, Aureobasidium pullulans, Phoma sp.1 and Ramichloridium sp., whereas the frequency of the endophyte Geniculosporium sp.1 varied with leaf age. The frequency of the epiphyte Cladosporium cladosporioides varied with both season and leaf age.     

Birch family and environmental conditions affect endophytic fungi in leaves

We investigated whether variation in foliar endophyte frequency among mountain birch trees from different maternal families was due in part to genetic differences among trees. The effect of different environmental conditions on the susceptibility of these mountain birch families to foliar endophytes was tested. The 3-year study was conducted in two tree gardens (altitudinal difference 180 m) with ten families of mountain birch. The frequency of the commonest endophytic fungus in mountain birch leaves, Fusicladium sp., was significantly affected by mountain birch family, with the ranking of families relative to Fusicladium sp. frequency being consistent from environment to environment. Variation in the frequency of Melanconium sp. was difficult to interpret because of significant family × garden × year interaction. Fusicladium sp. and Melanconium sp. endophytes were most frequent in different tree individuals, families and gardens. We conclude that mountain birch trees show heritable variation in their foliar endophyte frequency, and thus one of the conditions necessary for foliar endophytes of mountain birch trees to be able to affect the evolution of their host plant is fulfilled. However, the effect of mountain birch family on the frequency of endophytic fungi varies depending on the endophytic genera in question and partially also on environmental conditions. Received: 28 March 1998 / Accepted: 14 October 1998     

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.