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.     

Isolate-dependent impacts of fungal endophytes in a multitrophic interaction

Neotyphodium lolii , an endophytic fungus of perennial ryegrass, deters Argentine stem weevil, Listronotus bonariensis , an important insect pest of pastures in New Zealand. Deterrence is apparently due to several alkaloids the fungus produces. We asked if the fungus also affects the third trophic level. Specifically, we tested if several different isolates of the fungus altered the growth and survival of the parasitoid, Microctonus hyperodae . Adult weevils were collected from paddocks near Lincoln, New Zealand and maintained in an environmental chamber. Weevils were assorted into treatment groups and fed perennial ryegrass (cv. Nui) lacking endophyte infection (nil) or containing one of the following endophyte strains: ARW, AR1, AR6, AR37. All endophyte strains differed from one another with respect to the profile of alkaloids they produce. Following two weeks of acclimation, weevils were placed with M. hyperodae to allow for parasitization. Weevils were then placed into Petri dishes (10 per dish) and fed grass clippings from the appropriate treatment group. We collected weevils as they died and dissected them to assess the development of parasitoids. Emergence of prepupae from hosts and survival to adulthood were also recorded. Fungal isolates did not differ in their influence on weevil feeding or survival. In contrast, the effect of the endophyte on the parasitoid varied among isolates of the fungus. Isolates ARW and AR6 reduced parasitoid adult emergence relative to nil endophyte. In contrast, AR37 had no negative effect on survival of the parasitoid. Furthermore, an index of developmental rate showed that parasitoids developed more slowly when reared from hosts fed grass containing any of the strains of N. lolii , except AR37, compared to endophyte-free grass. Negative effects of the endophyte on parasitoid survival were associated with the presence of ergovaline while effects on parasitoid development rate were associated with the presence of any alkaloid.     

Symbiosis between grasses and asexual fungal endophytes

The symbiosis between vertically transmitted asexual endophytic fungi and grasses is common and generally considered to be mutualistic. Recent studies have accumulated evidence of negative effects of endophytes on plant fitness, prompting a debate on the true nature of the symbiosis. Genetic factors in each of the two partners show high variability and have a range of effects (from positive to negative) on plant fitness. In addition, interacting environmental factors might modify the nature of the symbiosis. Finally, competition and multitrophic interactions among grass consumers are influenced by endophytes, and the effects of plant neighbours or consumers could feedback to affect plant fitness. We propose a mutualism-parasitism continuum for the symbiosis between asexual endophytes and grasses, which is similar to the associations between plants and mycorrhizal fungi.     

Taxol from fungal endophytes and the issue of biodiversity

Fungi represent one of the most understudied and diverse group of organisms. Commonly, these organisms make associations with higher life forms and may proceed to biochemically mimic the host organism. An excellent example of this is the anticancer drug, taxol, which had been previously supposed to occur only in the plant genusTaxus (yew). However, taxol has been reported in a novel endophytic fungus—Taxomyces andreanae, but also has been demonstrated to occur in a number of unrelated fungal endophytes includingPestalotia, Pestalotiopsis, Fusarium, Alternaria, Pithomyces, Monochaetia and others. Thus, this report presents information on the presence of taxol among disparate fungal genera, and uses these observations as an additional argument to support efforts to study fungal endophytes and preserve their associated host plants.     

Antifungal metabolites from fungal endophytes of Pinus strobus

The extracts of five foliar fungal endophytes isolated from Pinus strobus (eastern white pine) that showed antifungal activity in disc diffusion assays were selected for further study. From these strains, the aliphatic polyketide compound 1 and three related sesquiterpenes 2-4 were isolated and characterized. Compound 2 is reported for the first time as a natural product and the E/Z conformational isomers 3 and 4 were hitherto unknown. Additionally, the three known macrolides; pyrenophorol (5), dihydropyrenophorin (6), and pyrenophorin (7) were isolated and identified. Their structures were elucidated by spectroscopic analyses including 2D NMR, HRMS and by comparison to literature data where available. The isolated compounds 1, 2, and 5 were antifungal against both the rust Microbotryum violaceum and Saccharomyces cerevisae.     

Bioactive natural products from marine sponges and fungal endophytes

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.     

The effects of fungal root endophytes on plant growth: a meta-analysis

Fungal root endophytes are plant associates that colonize root tissue internally without causing any obvious harm to their host. Although ubiquitous, this relationship is not well understood. Our objectives were to determine the effects of fungal root endophyte inoculation on plant biomass and nitrogen concentration by conducting an extensive meta-analysis. We also explored the effects of experimental conditions on the host–endophyte relationship. We performed analyses weighted with non-parametric variance on plant response to root endophytes from the Ascomycetes (excluding the Clavacipitaceae), including categorical analyses of 21 experimental factors, ranging from the identity of the host and the endophyte, to the composition of the growing medium. The response of total biomass to endophyte inoculation was 18 % lower than non-inoculated controls, while individually, root biomass, shoot biomass, and nitrogen concentration responses to endophyte inoculation were neutral. The identities of both the host and the endophyte had an influence, as did the original source of the endophyte (whether or not the isolate used originated from the same host species). Experimental conditions also influenced the plant–endophyte relationship, with the most important being the availability and sources of carbon and organic nitrogen, particularly peat moss. Although our analysis demonstrates that overall plant biomass and nitrogen concentration responses to ascomycetous root endophyte inoculation is neutral to negative, these results are somewhat confounded by among-study differences in experimental conditions, which undoubtedly contribute to the high levels of variability in plant response seen in the literature.     

Histological analyses of the fungal endophytes in Rosa hybrida

Endophytes are fungi that cause asymptomatic infections in leaves and stems of healthy plants. This study evaluated the presence of endophytic fungi on leaves of Rosa hybrida collected in Bogota, (Colombia) using histological techniques and light microscopy. Histological preparations showed fungal mycelium with few cells in the vascular tissue. Colonization of mesophyll cells was not observed. Visualization of fungal cells within plant tissues is a confirmatory test of the endophytic habitat of fungi isolated from leaves of Rosa hybrida.     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

Molecular phylogeny and paclitaxel screening of fungal endophytes from Taxus globosa

We studied the endophytic mycoflora associated with Taxus globosa, the Mexican yew. The study localities; Las Avispas (LA), San Gaspar (SG), and La Mina (LM) were three segments of cloud forest within the range of Sierra Gorda Biosphere Reserve, México. Overall, 245 endophytes were isolated and 105 representative Ascomycota (morphotaxons) were chosen for phylogenetic and genotypic characterization. Maximum likelihood analyses of large subunit of ribosomal RNA (LSU) rDNA showed well-supported clades of Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes. Analyses of ITS rDNA groups showed 57 genotypes (95% sequence similarity), in general consistent with the phylogenetically delimitated taxa based on LSU rDNA sequences. The endophyte diversity measured by Fisher's α, Shanonn, and Simpson indices was ca. three-fold and ca. two-fold greater in LM than in LA and SG respectively. A screening for paclitaxel using a competitive inhibition enzyme immunoassay showed 16 positive isolates producing between 65 and 250 ng l(-1). The isolates included Acremonium, Botryosphaeria, Fusarium, Gyromitra, Nigrospora, Penicillium, three novel Pleosporales, and Xylaria.     

Balancing multiple mutualists: asymmetric interactions among plants, arbuscular mycorrhizal fungi, and fungal endophytes

Most organisms engage in beneficial interactions with other species; however, little is known regarding how individuals balance the competing demands of multiple mutualisms. Here we examine three-way interactions among a widespread grass, Schedonorus phoenix , a protective fungal endophyte aboveground, Neotyphodium coenophialum , and nutritional symbionts (arbuscular mycorrhizal fungi) belowground. In a greenhouse experiment, we manipulated the presence/absence of both fungi and applied a fertilizer treatment to individual plants. Endophyte presence in host plants strongly reduced mycorrhizal colonization of roots. Additionally, for plants with the endophyte, the density of endophyte hyphae was negatively correlated with mycorrhizal colonization, suggesting a novel role for endophyte abundance in the interaction between the symbionts. Endophyte presence increased plant biomass, and there was a positive correlation between endophyte hyphal density and plant biomass. The effects of mutualists were asymmetric: mycorrhizal fungi treatments had no significant impact on the endophyte and negligible effects on plant biomass. Fertilization affected all three species – increasing plant biomass and endophyte density, but diminishing mycorrhizal colonization. Mechanisms driving negative effects of endophytes on mycorrhizae may include inhibition via endophyte alkaloids, altered nutritional requirements of the host plant, and/or temporal and spatial priority effects in the interactions among plants and multiple symbionts.     

Root fungal endophytes: identity,phylogeny and roles in plant tolerance to metal stress

Metal trace elements accumulate in soils mainly because of anthropic activities, leading living organisms to develop strategies to handle metal toxicity. Plants often associate with root endophytic fungi, including nonmycorrhizal fungi, and some of these organisms are associated with metal tolerance. The lack of synthetic analyses of plant-endophyte-metal tripartite systems and the scant consideration for taxonomy led to this review aiming (1) to inventory non-mycorrhizal root fungal endophytes described with respect to their taxonomic diversity and (2) to determine the mutualistic roles of these plant-fungus associations under metal stress. More than 1500 species in 100 orders (mainly Hypocreales and Pleosporales) were reported from a wide variety of environments and hosts. Most reported endophytes had a positive effect on their host under metal stress, but with various effects on metal uptake or translocation and no clear taxonomic consistency. Future research considering the functional patterns and dynamics of these associations is thus encouraged.     

Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae)

Roots of plants in the genus Enkianthus, which belongs to the earliest diverging lineage in the Ericaceae, are commonly colonized by arbuscular mycorrhizal (AM) fungi. We documented the community of fungal root endophytes associated with Enkianthus species using a culture-based method for better understanding the members of root-colonizing fungi, except for AM fungi. Fungal isolates were successfully obtained from 610 out of 3,599 (16.9 %) root segments. Molecular analysis of fungal cultures based on ribosomal internal transcribed spacer (ITS) sequences yielded 63 operational taxonomical units (OTUs: 97 % sequence similarity cutoff) from 315 representative isolates. Further phylogenetic analysis showed that most (296 isolates) belonged to Ascomycota and were either members of Helotiales (Dermataceae, Hyaloscyphaceae, Phialocephala and Rhizoscyphus ericae aggregate), Oidiodendron, or other Pezizomycotina. Twenty-three out of 63 OTUs, which mainly consisted of Leotiomycetes, showed high similarities with reference sequences derived from roots of other ericaceous plants such as Rhododendron. The results indicated that Enkianthus houses variable root mycobionts including putative endophytic and mycorrhizal fungi in addition to AM fungi.     

Characterization of cellulases of fungal endophytes isolated from Espeletia spp.

Endophytes are microorganisms that asymptomatically invade plant tissues. They can stimulate plant growth and/or provide defense against pathogen attacks through the production of secondary metabolites. Most endophyte species are still unknown, and because they may have several applications, the study of their metabolic capabilities is essential. We characterized 100 endophytes isolated from Espeletia spp., a genus unique to the paramo ecosystem, an extreme environment in the Andean mountain range. We evaluated the cellulolytic potential of these endophytes on the saccharification of the oil palm empty fruit bunch (OPEFB). The total cellulolytic activity was measured for each endophyte on filter paper (FPA). In addition, the specific carboxymethyl cellulase (CMCase), exoglucanase, and β-glucosidase activities were determined. We found four fungi positive for cellulases. Of these fungi, Penicillium glabrum had the highest cellulolytic activity after partial purification, with maximal CMCase, exoglucanase and β-glucosidase enzyme activities of 44.5, 48.3, and 0.45 U/ml, respectively. Our data showed that the bioprospection of fungi and the characterization of their enzymes may facilitate the process of biofuel production.     

Consequences of simultaneous interactions of fungal endophytes and arbuscular mycorrhizal fungi with a shared host grass

Plants simultaneously associate with multiple microbial symbionts throughout their lifetimes. To address the question of whether the effects of simultaneous symbionts are contingent on the specific identities, we conducted a greenhouse experiment manipulating the presence and identities of arbuscular mycorrhizal fungi (AMF) and fungal endophytes on the shared host grass Elymus hystrix. Each plant host was inoculated with one of two AMF species having varying effects on host growth, or a sterile soil control. Further, we used naturally occurring endophyte‐infected (E+) and uninfected (E–) individuals from two populations of the endophyte Epichloë elymi that varied in their interaction with E. hystrix. We then measured responses of plants, AMF, and fungal endophytes. Overall, we found that the combined effects of AMF and fungal endophytes on plant growth were additive, reflecting the mutualistic quality of each symbiont independently interacting with host plants. However, fungal endophyte infection differentially altered hyphal colonization of the two AMF species and the identity of the coinfecting AMF species affected fungal endophyte fitness traits. The results of this study demonstrate that the outcome of interspecific symbiotic interactions varies with partner identity such that the effects of simultaneous symbioses can not be generalized.