Fungal endophyte effects on invasive Phragmites australis performance in field and growth chamber environments

Manipulating plant microbiomes may provide control of invasive species. Invasive Phragmites australis has spread rapidly in North American wetlands, causing significant declines in native biodiversity. To test microbiome effects on host growth, we inoculated four common fungal endophytes into replicated Phragmites genotypes and monitored their growth in field and growth chamber environments. Inoculations were highly successful in the growth chamber but inoculated plants in the field were rapidly colonized by diverse endophytes from the local environment. There were significant genotype effects and minimal inoculation effects in both experiments with a significant inoculation × genotype interaction on tiller height in the field. Our results demonstrate that endophyte inoculation treatments are feasible, but repeated inoculations may be required to maintain high titer in plants subject to endophyte colonization from the local environment. Future studies should investigate a wider range of fungal endophytes to identify taxa that inhibit Phragmites and other invaders.     

The interactive effects of plant microbial symbionts: a review and meta-analysis

In nature, plants often associate with multiple symbionts concurrently, yet the effects of tripartite symbioses are not well understood. We expected synergistic growth responses from plants associating with functionally distinct symbionts. In contrast, symbionts providing similar benefits to a host may reduce host plant growth. We reviewed studies investigating the effect of multiple interactions on host plant performance. Additionally, we conducted a meta-analysis on the studies that performed controlled manipulations of the presence of two microbial symbionts. Using response ratios, we investigated the effects on plants of pairs of symbionts (mycorrhizal fungi, fungal endophytes, and nitrogen-fixers). The results did not support the view that arbuscular mycorrhizal (AM) fungi and rhizobia should interact synergistically. In contrast, we found the joint effects of fungal endophytes and arbuscular mycorrhizal fungi to be greater than expected given their independent effects. This increase in plant performance only held for antagonistic endophytes, whose negative effects were alleviated when in association with AM fungi, while the impact of beneficial endophytes was not altered by infection with AM fungi. Generalizations from the meta-analysis were limited by the substantial variation within types of interactions and the data available, highlighting the need for more research on a range of plant systems.     

A suite of rare microbes interacts with a dominant,heritable, fungal endophyte to influence plant trait expression

Endophytes are microbes that live, for at least a portion of their life history, within plant tissues. Endophyte assemblages are often composed of a few abundant taxa and many infrequently observed, low-biomass taxa that are, in a word, rare. The ways in which most endophytes affect host phenotype are unknown; however, certain dominant endophytes can influence plants in ecologically meaningful ways—including by affecting growth and immune system functioning. In contrast, the effects of rare endophytes on their hosts have been unexplored, including how rare endophytes might interact with abundant endophytes to shape plant phenotype. Here, we manipulate both the suite of rare foliar endophytes (including both fungi and bacteria) and Alternaria fulva–a vertically transmitted and usually abundant fungus–within the fabaceous forb Astragalus lentiginosus. We report that rare, low-biomass endophytes affected host size and foliar %N, but only when the heritable fungal endophyte (A. fulva) was not present. A. fulva also reduced plant size and %N, but these deleterious effects on the host could be offset by a negative association we observed between this heritable fungus and a foliar pathogen. These results demonstrate how interactions among endophytic taxa determine the net effects on host plants and suggest that the myriad rare endophytes within plant leaves may be more than a collection of uninfluential, commensal organisms, but instead have meaningful ecological roles.Subject terms: Microbial ecology, Community ecology, Microbial ecology     

An ecological framework for understanding the roles of Epichloë endophytes on plant defenses against fungal diseases

Plants harbor a wide diversity of microorganisms in their tissues. Some of them have a long co-evolutionary history with their hosts, likely playing a pivotal role in regulating the plant interaction with other microbes such as pathogens. Some cool-season grasses are symbiotic with Epichloë fungal endophytes that grow symptomless and systemically in aboveground tissues. Among the many benefits that have been ascribed to endophytes, their role in mediating plant interactions with pathogens has been scarcely developed. Here, we explored the effects of Epichloë fungal endophytes on the interaction of host grasses with fungal pathogens. We made a meta-analysis that covered a total of 18 host grass species, 11 fungal endophyte species, and 22 fungal pathogen species. We observed endophyte-mediated negative effects on pathogens in vitro and in planta. Endophyte negative effects on pathogens were apparent not only in laboratory but also in greenhouse and field experiments. Epichloë fungal endophytes had negative effects on pathogen growth and spores' germination. On living plants, endophytes reduced both severity and incidence of the disease as well as colonization and subsequent infection of seeds. Symbiosis with endophytes showed an inhibitory effect on debilitator and killer pathogens, but not on castrators, and this effect did not differ among biotrophic or necrotrophic lifestyles. We found that this protection can be direct through the production of fungistatic compounds, the competition for a common resource, or the induction of plant defenses, and indirect associated with endophyte-generated changes in the abiotic or the biotic environment. Several mechanisms operate simultaneously and contribute differentially to the reduction of disease within grass populations.     

Fungal root endophytes of the carnivorous plant Drosera rotundifolia

As carnivorous plants acquire substantial amounts of nutrients from the digestion of their prey, mycorrhizal associations are considered to be redundant; however, fungal root endophytes have rarely been examined. As endophytic fungi can have profound impacts on plant communities, we aim to determine the extent of fungal root colonisation of the carnivorous plant Drosera rotundifolia at two points in the growing season (spring and summer). We have used a culture-dependent method to isolate fungal endophytes and diagnostic polymerase chain reaction methods to determine arbuscular mycorrhizal fungi colonisation. All of the roots sampled contained culturable fungal root endophytes; additionally, we have provided molecular evidence that they also host arbuscular mycorrhizal fungi. Colonisation showed seasonal differences: Roots in the spring were colonised by Articulospora tetracladia, two isolates of uncultured ectomycorrhizal fungi, an unidentified species of fungal endophyte and Trichoderma viride, which was present in every plant sampled. In contrast, roots in the summer were colonised by Alatospora acuminata, an uncultured ectomycorrhizal fungus, Penicillium pinophilum and an uncultured fungal clone. Although the functional roles of fungal endophytes of D. rotundifolia are unknown, colonisation may (a) confer abiotic stress tolerance, (b) facilitate the acquisition of scarce nutrients particularly at the beginning of the growing season or (c) play a role in nutrient signalling between root and shoot.     

Diversity and host preference of fungi co-inhabiting Cenococcum mycorrhizae

Diverse fungal assemblages colonize the fine feeder roots of woody plants, including mycorrhizal fungi, fungal root endophytes and soil saprotrophs. The fungi co-inhabiting Cenococcum geophilum ectomycorrhizae (ECM) of Abies balsamea, Betula papyrifera and Picea glauca were studied at two boreal forest sites in Eastern Canada by direct PCR of ITS rDNA. 50 non-Cenococcum fungal sequence types were detected, including several potentially mycorrhizal species as well as fungal root endophytes. Non-melanized ascomycetes dominated, in contrast to the dark septate endophytes (DSE) reported in most culture dependent studies. The results demonstrate significant differences in root associated fungal assemblages among the host species studied. Fungal diversity was also host dependent, with P. glauca roots supporting a more diverse community than A. balsamea. Differences in root associated fungal communities may well influence ecological interactions among host plant species.     

Multitrophic interactions among fungal endophytes,bees, and <Emphasis Type="Italic">Baccharis dracunculifolia</Emphasis>: resin tapering for propolis production leads to endophyte infection

The tropics are known for their high diversity of plants, animals, and biotic interactions, but the role of the speciose endophytic fungi in these interactions has been mostly neglected. We report a unique interaction among plant sex, bees, and endophytes on the dioecious shrub, Baccharis dracunculifolia (Asteraceae). We assessed whether there was an association between resin collection by bees and fungal endophytes considering the host plant sex. We hypothesized that resin collection by the Africanized honey bee, Apis mellifera L. (Apidae) could favor the entry of endophytes in B. dracunculifolia. Specifically, we tested the hypotheses that (1) bees damage the leaf buds of female and male plant at different proportions; (2) damage on leaf buds increases the richness of endophytic fungi; (3) endophyte richness differs between female and male plants; and (4) in vitro growth of endophytes depends on the sex of the plant individual from which the resin was extracted. Endophyte richness and proportion of leaf bud damage did not vary between the plant sexes. However, species similarity of endophytes between female and male plants was 0.33. Undamaged leaf buds did not show culturable endophytes, with all fungi exclusively found in damaged leaf buds. Endophyte composition changed with the plant sex. The endophytes exclusively found in female plants did not develop in the presence of male resin extract. These findings highlight that resin collection by A. mellifera for propolis production favors the entry of endophytic fungi in B. dracunculifolia. Additionally, endophyte composition and growth are influenced by plant sex.     

Antimicrobial fungal endophytes from the botanical medicine goldenseal (Hydrastis canadensis)

The potential of fungal endophytes to alter or contribute to plant chemistry and biology has been the topic of a great deal of recent interest. For plants that are used medicinally, it has been proposed that endophytes might play an important role in biological activity. With this study, we sought to identify antimicrobial fungal endophytes from the medicinal plant goldenseal (Hydrastis canadensis L., Ranunculaceae), a plant used in traditional medicine to treat infection. A total of 23 fungal cultures were obtained from surface-sterilized samples of H. canadensis roots, leaves and seeds. Eleven secondary metabolites were isolated from these fungal endophytes, five of which had reported antimicrobial activity. Hydrastis canadensis plant material was then analyzed for the presence of fungal metabolites using liquid chromatography coupled to high resolving power mass spectrometry. The antimicrobial compound alternariol monomethyl ether was detected both as a metabolite of the fungal endophyte Alternaria spp. isolated from H. canadensis seeds, and as a component of an extract from the H. canadensis seed material. Notably, fungi of the Alternaria genus were isolated from three separate accessions of H. canadensis plant material collected in a time period spanning 5 years. The concentration of alternariol monomethyl ether (991 mg/kg in dry seed material) was in a similar range to that previously reported for metabolites of ecologically important fungal endophytes. The seed extracts themselves, however, did not possess antimicrobial activity.     

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.     

Culturable Endophytes Associated with Soybean Seeds and Their Potential for Suppressing Seed-Borne Pathogens

Seed-borne pathogens in crops reduce the seed germination rate and hamper seedling growth, leading to significant yield loss. Due to the growing concerns about environmental damage and the development of resistance to agrochemicals among pathogen populations, there is a strong demand for eco-friendly alternatives to synthetic chemicals in agriculture. It has been well established during the last few decades that plant seeds harbor diverse microbes, some of which are vertically transmitted and important for plant health and productivity. In this study, we isolated culturable endophytic bacteria and fungi from soybean seeds and evaluated their antagonistic activities against common bacterial and fungal seed-borne pathogens of soybean. A total of 87 bacterial isolates and 66 fungal isolates were obtained. Sequencing of 16S rDNA and internal transcribed spacer amplicon showed that these isolates correspond to 30 and 15 different species of bacteria and fungi, respectively. Our antibacterial and antifungal activity assay showed that four fungal species and nine bacterial species have the potential to suppress the growth of at least one seed-borne pathogen tested in the study. Among them, Pseudomonas koreensis appears to have strong antagonistic activities across all the pathogens. Our collection of soybean seed endophytes would be a valuable resource not only for studying biology and ecology of seed endophytes but also for practical deployment of seed endophytes toward crop protection.     

Root-associated community composition and co-occurrence patterns of fungi in wild grapevine

Crops’ wild relatives host a wide range of microorganisms, including some beneficial species that are not found or are under-represented in the domesticated crops. Our goal was to study the underexplored composition of root-associated fungal communities in endangered wild grapevines. We found high taxonomic diversity representing multiple trophic guilds that include beneficial symbiotrophs and endophytes. Soil factors explain a relatively small part of their overall variability. In contrast, the majority of the associated fungal taxa shows a close fit to the neutral model for prediction of their distributions. Only beneficial arbuscular mycorrhizal fungi and the pathogenic Ilyonectria depart from the neutral distribution model and form intimate interactions with the plant host. In addition, pathogenic fungi rarely occurred in samples that included ectomycorrhizal fungi, which suggested potentially applicable inter-microorganism interactions. High abundance and diversity of fungal endophytes on the wild grapevine roots highlight the need for their careful consideration in future studies.     

A Phylogenetic Evaluation of Whether Endophytes Become Saprotrophs at Host Senescence

Fungal endophytes and saprotrophs generally play an important ecological role within plant tissues and dead plant material. Several reports based solely on morphological observations have postulated that there is an intimate link between endophytes and saprotrophs. This study aims to provide valuable insight as to whether some endophytic fungi manifest themselves as saprotrophs upon host decay. Ribosomal DNA-based sequence comparison and phylogenetic relationships from 99 fungal isolates (endophytes, mycelia sterilia, and saprotrophs) recovered from leaves and twigs of Magnolia liliifera were investigated in this study. Molecular data suggest there are fungal taxa that possibly exist as endophytes and saprotrophs. Isolates of Colletotrichum, Fusarium, Guignardia, and Phomopsis, which are common plant endophytes, have high sequence similarity and are phylogenetically related to their saprotrophic counterparts. This provides evidence to suggest that some endophytic species change their ecological strategies and adopt a saprotrophic lifestyle. The implication of these findings on fungal biodiversity and host specificity is also discussed.     

Root endophytic fungi show low levels of interspecific competition in planta

Roots are associated with fungal communities that affect plant growth and health. Individual root-associated fungi have different effects on plant performance, from detrimental to beneficial, but it is barely known how their inter-species interactions determine plant fitness. Here, we evaluate in planta interactions among dominant root-colonizing fungi with different degrees of phylogenetic and trait similarity, and the impact of their co-occurrence on their respective ability to colonize roots and their effects on plant growth. An in vitro bioassay with Arabidopsis thaliana as host plant was used for the co-cultivation with individual or paired combinations of fungal strains. Root colonization by strains was monitored using real-time quantitative PCR, and the effects on their host's growth were estimated by measuring plant biomass. Strains had variable effects on plant growth, although these effects were mostly modest and little affected by the presence of other fungi. Abundance of each fungus in roots responded differently to co-inoculation, but competition between strains was not associated with their similarity in functional traits. Our findings show little competition between dominant fungal root endophytes, which suggests that they occupy complementary root niches and could explain the high fungal diversity colonizing healthy hosts in natural conditions.     

Endophytic fungi from the root tubers of medicinal plant Stephania dielsiana and their antimicrobial activity

The adverse effects of chemical synthetic fungicides on agricultural fields and the environment are driving a need to search for safer and less environmentally harmful plant protectants to move toward more sustainable development of agriculture. The endophytic fungal community associated with the medicinal plant Stephania dielsiana, and its potential for providing antimicrobial secondary metabolites were investigated. A total of 26 isolates of endophytic fungi were obtained, and 21 isolates were identified and classified into eight different genera, including Briansuttonomyces, Glomerella, Pleosporales, Diaporthe, Phoma, Penicillium, Periconia and Colletotrichum, and the most frequent endophytic species obtained were Diaporthe phaseolorum, Penicillium sp., Periconia igniari and Colletotrichum sp. The ethyl acetate (EtOAc) extract of the endophytic fungus Diaporthe phaseolorum Stdif6 displayed the most significant antifungal activity against all tested phytopathogens, with EC₅₀ values ranging from 0.0138 to 0.3103 mg/mL. While the EtOAc extract of the endophytic fungus Penicillium sp. Stdif9 exhibited greater potential for antibacterial activity, with the minimum inhibitory concentration (MIC) values against seven bacteria ranging from 1.25 to 6 mg/mL. The remarkable antimicrobial activity of fungal endophytes suggests that fungal endophytes harbored inside the root tubers of S. dielsiana hold great promise as biocontrol agents against a broad spectrum of economically significant pathogens.     

The endophytic fungal communities associated with the leaves and roots of the common reed (Phragmites australis) in Lake Trasimeno (Perugia,Italy) in declining and healthy stands

We surveyed the fungal endophytes in the leaves and roots of Phragmites australis plants along a gradient of reed decline at Lake Trasimeno (central Italy) in Oct. 2010. An integrated approach consisting of cultivation and molecular identification was used. Endophytes were recovered from 61.59 % of the samples, with a total of 1 541 isolates. On the basis of a molecular analysis of the rDNA Internal Transcribed Spacer (ITS) region, 25 operational taxonomic units (OTUs) were identified. Fusarium sp. (OTU 21) and Gibberella moniliformis (OTU 1) were the most frequently isolated fungi. Comparisons of the leaf and root samples demonstrated spatial heterogeneity in the endophyte assemblages among the plant parts and sites. In this study, we have shown that reed plants in different states of decline harbour different endophytic communities. This finding may help to understand the very complex scenario of reed die-back.     

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

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

内生真菌感染对宿主羊草抗病性的影响

以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichlobromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C.lunata和C.sp.侵染后的病斑数或病斑长度,但对B.sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E.bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。     

Novel Symbiotic Protoplasts Formed by Endophytic Fungi Explain Their Hidden Existence,Lifestyle Switching,and Diversity within the Plant Kingdom

Diverse fungi live all or part of their life cycle inside plants as asymptomatic endophytes. While endophytic fungi are increasingly recognized as significant components of plant fitness, it is unclear how they interact with plant cells; why they occur throughout the fungal kingdom; and why they are associated with most fungal lifestyles. Here we evaluate the diversity of endophytic fungi that are able to form novel protoplasts called mycosomes. We found that mycosomes cultured from plants and phylogenetically diverse endophytic fungi have common morphological characteristics, express similar developmental patterns, and can revert back to the free-living walled state. Observed with electron microscopy, mycosome ontogeny within Aureobasidium pullulans may involve two organelles: double membrane-bounded promycosome organelles (PMOs) that form mycosomes, and multivesicular bodies that may form plastid-infecting vesicles. Cultured mycosomes also contain a double membrane-bounded organelle, which may be homologous to the A. pullulans PMO. The mycosome PMO is often expressed as a vacuole-like organelle, which alternatively may contain a lipoid body or a starch grain. Mycosome reversion to walled cells occurs within the PMO, and by budding from lipid or starch-containing mycosomes. Mycosomes discovered in chicken egg yolk provided a plant-independent source for analysis: they formed typical protoplast stages, contained fungal ITS sequences and reverted to walled cells, suggesting mycosome symbiosis with animals as well as plants. Our results suggest that diverse endophytic fungi express a novel protoplast phase that can explain their hidden existence, lifestyle switching, and diversity within the plant kingdom. Importantly, our findings outline “what, where, when and how”, opening the way for cell and organelle-specific tests using in situ DNA hybridization and fluorescent labels. We discuss developmental, ecological and evolutionary contexts that provide a robust framework for continued tests of the mycosome phase hypothesis.     

The potential saprotrophic capacity of foliar endophytic fungi from Quercus gambelii

Endophytic fungi occur in living tissues of terrestrial plants. Many of these fungi are primarily biotrophic, but the trophic range of endophytic fungi as a group may not be fully appreciated. In this study, our goals were (1) for the Class 3 foliar endophytic fungi isolated from Quercus gambelii, determine their potential saprotrophic capacity, which we define as the difference in growth rate in culture on Quercus gambelii leaf litter medium and control medium lacking leaf litter and (2) quantify sources of variation among isolates of these endophytic fungi in potential saprotrophic capacity, including variation due to microsite within host trees (leaves receiving full sun vs. shade) and variation within and among fungal genera. We found that 48 of the 49 tested endophytic fungal isolates have significant potential saprotrophic capacity. Contrary to expectation, the amount of solar radiation available to the leaf from which the fungi were isolated had no significant impact on potential saprotrophic capacity and there was more variability in potential saprotrophic capacity among isolates within a genus than among genera. Our results suggest that some Class 3 endophytic fungi may have the potential to function as saprotrophic fungi within plant litter, but this remains to be seen for these Quercus gambelii isolates under more natural circumstances.