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

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.     

Endophyte inoculation enhances Ulmus minor resistance to Dutch elm disease

Certain fungal endophytes improve plant resistance to biotic stresses in forest trees. In this study, three stem fungal endophytes belonging to classes Cystobasidiomycetes, Eurotiomycetes and Dothideomycetes were selected from 210 isolates for their potential as enhancers of Ulmus minor resistance to Ophiostoma novo-ulmi. We evaluated phenotypic traits of these endophytes that could be beneficial for inhibiting O. novo-ulmi in the host plant. Under in vitro conditions, the Dothideomycetous isolate YCB36 strongly inhibited O. novo-ulmi growth, released antipathogenic VOCs, chitinases and siderophores, and overlapped with the pathogen in nutrient utilization patterns. These functional traits could explain the 40% reduction in leaf wilting due to O. novo-ulmi in elm trees pre-inoculated with this endophyte. Ulmus minor trees inoculated with this endophyte showed increased leaf stomatal conductance and higher concentrations of flavonoids and total phenolic compounds in xylem tissues, suggesting induction of defence metabolism.     

The influence of genetics,defensive chemistry and the fungal microbiome on disease outcome in whitebark pine trees

The invasive fungal pathogen Cronartium ribicola infects and kills whitebark pine (Pinus albicaulis) throughout western North America. Whitebark pine has been proposed for listing under the Endangered Species Act in the USA, and the loss of this species is predicted to have severe impacts on ecosystem composition and function in high‐elevation forests. Numerous fungal endophytes live inside whitebark pine tissues and may influence the severity of C. ribicola infection, either directly by inhibition of pathogen growth or indirectly by the induction of chemical defensive pathways in the tree. Terpenes, a form of chemical defence in pine trees, can also influence disease. In this study, we characterized fungal endophyte communities in whitebark pine seedlings before and after experimental inoculation with C. ribicola, monitored disease progression and compared fungal community composition in susceptible vs. resistant seedlings in a common garden. We analysed the terpene composition of these same seedlings. Seed family identity or maternal genetics influenced both terpenes and endophyte communities. Terpene and endophyte composition correlated with disease severity, and terpene concentrations differed in resistant vs. susceptible seedlings. These results suggest that the resistance to C. ribicola observed in natural whitebark pine populations is caused by the combined effects of genetics, endophytes and terpenes within needle tissue, in which initial interactions between microbes and hosts take place. Tree genotype, terpene and microbiome combinations associated with healthy trees could help to predict or reduce disease severity and improve outcomes of future tree breeding programmes.     

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).     

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.     

Genetic Diversity and Structure of Neotyphodium Species and Their Host Achnatherum sibiricum in a Natural Grass–Endophyte System

Achnatherum sibiricum (Poaceae) is a perennial bunchgrass native to the Inner Mongolia Steppe of China. This grass is commonly infected by epichloë endophytes with high-infection frequencies. Previously, we identified two predominant Neotyphodium spp., N. sibiricum and N. gansuense. In the present study, genetic diversity and structure were analyzed for the two predominant Neotyphodium spp. as well as the host grass. We obtained 103 fungal isolates from five populations; 33 were identified as N. sibiricum and 61 as N. gansuense. All populations hosted both endophytic species, but genetic variation was much higher for N. gansuense than for N. sibiricum. The majority of fungal isolates were haploid, and 13% of them were heterozygous at one SSR locus, suggesting hybrid origins of those isolates. Significant linkage disequilibrium of fungal SSR loci suggested that both fungal species primarily propagate by clonal growth through plant seeds, whereas variation in genetic diversity and the presence of hybrids in both endophytic species revealed that although clonal propagation was prevalent, occasional recombination might also occur. By comparing genetic differentiation among populations, we found around 4–7-fold greater differentiation of endophyte populations than host populations, implying more restricted gene flow of endophytes than hosts. We proposed that endophyte infection of A. sibiricum might confer the host some selective advantages under certain conditions, which could help to maintain high-endophyte-infection frequencies in host populations, even when their gene flows do not match each other. Furthermore, we suggested that the same genotype of endophyte as well as host should be confirmed if the objective of the study is to know the influence of endophyte or host genotype on their symbiotic relationship, instead of just considering whether the plant is infected by an endophyte or not, since endophytes from the same host species could exhibit high levels of genetic diversity, which is likely to influence the outcome of their symbiotic relationship.     

Fungal communities associated with species of Fraxinus tolerant to ash dieback,and their potential for biological control

Ash dieback, caused by the fungus Hymenoscyphus fraxineus, has threatened ash trees in Europe for more than two decades. However, little is known of how endophytic communities affect the pathogen, and no effective disease management tools are available. While European ash (Fraxinus excelsior) is severely affected by the disease, other more distantly related ash species do not seem to be affected. We hypothesise that fungal endophytic communities of tolerant ash species can protect the species against ash dieback, and that selected endophytes have potential as biocontrol agents. These hypotheses were tested by isolating members of the fungal communities of five tolerant ash species, and identifying them using ITS regions. Candidate endophytes were tested by an in vitro antagonistic assay with H.fraxineus. From a total of 196 isolates we identified 9 fungal orders, 15 families, and 40 species. Fungi in orders Pleosporales, such as Boeremia exigua and Diaporthe spp., and Hypocreales (e.g., Fusarium sp.), were recovered in most communities, suggesting they are common taxa. The in vitro antagonistic assay revealed five species with high antagonistic activity against H. fraxineus. These endophytes were identified based on ITS region as Sclerostagonospora sp., Setomelanomma holmii, Epicoccum nigrum, B. exigua and Fusarium sp. Three of these taxa have been described previously as antagonists of plant pathogenic microbes, and are of interest for future studies of their potential as biological control agents against ash dieback, especially for valuable ash trees in parks and urban areas.     

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.     

Endophytic fungi reduce leaf-cutting ant damage to seedlings

Our study examines how the mutualism between Atta colombica leaf-cutting ants and their cultivated fungus is influenced by the presence of diverse foliar endophytic fungi (endophytes) at high densities in tropical leaf tissues. We conducted laboratory choice trials in which ant colonies chose between Cordia alliodora seedlings with high (E_high) or low (E_low) densities of endophytes. The E_high seedlings contained 5.5 times higher endophyte content and a greater diversity of fungal morphospecies than the E_low treatment, and endophyte content was not correlated with leaf toughness or thickness. Leaf-cutting ants cut over 2.5 times the leaf area from E_low relative to E_high seedlings and had a tendency to recruit more ants to E_low plants. Our findings suggest that leaf-cutting ants may incur costs from cutting and processing leaves with high endophyte loads, which could impact Neotropical forests by causing variable damage rates within plant communities.     

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     

Acremonium as an endophytic bioagent against date palm Fusarium wilt

A total of 250 endophytic fungal isolates, representing 30 morphotaxa, were isolated and characterised, they were collected from the different living symptomless parts of date palm trees of orchards of six Egyptian governorates. Colonisation was greater in samples from the midrib than in those from laminar tissue and slightly greater at the tip of the lamina compared with the base of the leaf. Acremonium spp. were frequently isolated as date palm root endophytes. Acremonium isolates were screened in Petri dishes to select the highest antagonistic one against an Algerian isolate of Fusarium oxysporum f.sp. albedinis. Two-week-old axenically reared date palm seedlings grown in Petri dishes were directly injected with spore suspension (1.5?×?10⁷ spores/ml) of a pure culture of the virulent antagonistic isolate of Acremonium sp. One week after endophytic colonisation, date palm seedlings were then challenged with the pathogen, Fusarium albedinis. The challenged seedlings exhibited a significant reduction in wilt symptom percentage (by 87.0%), while the seedlings exposed to Fusarial toxin without pathogen exhibited the wilt disease symptoms. This indicates that the endophyte ably depresses any toxic action of F. albedinis. The endophytic fungus was recovered from sites distant from the point of inoculation after six?months from the application, indicating that the Acremonium sp. has the potential to move throughout the tissue plant, even the end time of trial. The Acremonium mode of action, as a biocontrol agent, was discussed.     

Characteristics of foliar fungal endophyte assemblages and host effective components in Salvia miltiorrhiza Bunge

Salvia miltiorrhiza Bunge, a well-known medicinal plant, has more than 20 effective components. The aim of this study was to comprehensively investigate foliar fungal endophyte communities of S. miltiorrhiza and explore the inside relationship between host-specific fungal endophytes and effective components accumulation. Five plant samples were collected from four geological different provinces in China. Foliar fungal endophyte communities were determined by terminal restriction fragment length polymorphism (T-RFLP) of the ITS region. Effective components were analyzed with high-performance liquid chromatography. The results showed that S. miltiorrhiza foliage harbored a large diversity of fungal endophytes. Principal component analysis revealed similar T-RFLP profiles and the characteristics of the 24 effective components among the five samples, which could be clustered into three groups. In foliar T-RFLP profiles derived from the restriction digestion by CfoI, HaeIII, MspI, or TaqI, there were identical 45, 42, 38, and 34 terminal restriction fragments (T-RFs) from the five samples. We consider these T-RFs as host-specific fungal endophytes. Correlation analysis of these T-RFs’ area and 24 effective components contents revealed a significant correlationship between some host-specific fungal endophytes and foliage or root effective components accumulation.     

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。     

Role of foliar fungal endophytes in litter decomposition among species and population origins

Litter decomposition is a key ecosystem process which returns nutrients from dead plant material to mineral forms in the soil. We examined whether systemic fungal endophytes modulate recycling of nutrients directly by altering litter decomposition. We studied litter decomposition mediated by Epichloë endophytes in litter-bag experiments. We examined direct endophyte effects on litter decomposition in wild populations and cultivars of Schedonorus phoenix and Schedonorus pratensis. In the first experiment, endophyte presence tended to increase litter decomposition rate in cultivars of the two grass species (S. phoenix and S. pratensis). However, in the second experiment plant origin had a stronger influence than endophyte symbiosis in S. phoenix. Interestingly, the initial level of alkaloids was associated positively with decomposition in S. phoenix populations. Characteristics associated with litter quality were not clearly related to either endophytes or decomposition rate. Our results suggest that endophytes can enhance litter breakdown but their role in nutrient cycling is far more complex depending on plant population origin.     

Resistance to Dutch Elm Disease Reduces Presence of Xylem Endophytic Fungi in Elms (Ulmus spp.)

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.     

Fungal endophytes and phytochemistry of oak foliage: determinants of oviposition preference of leafminers?

Sedentary insect herbivores, such as gallformers and leafminers, are usually non-randomly distributed among and within host plants. Dispersion of these insects is largely a function of female oviposition choice. In field experiments and observations spanning two growing seasons, we tested the hypothesis that selective oviposition on individual leaves within trees by the dominant herbivore of Emory oak, the monophagous leaf-miner Cameraria sp. nov., is determined by the probability of colonization by endophytic fungi. These fungi are alleged to act as plant mutualists by deterring, killing, or inhibiting the growth of insect herbivores. We found that leaves selected by females for oviposition and paired, unmined leaves were equally likely to be colonized by fungal endophytes. Furthermore, condensed and hydrolyzable tannin levels, purported inhibitors of fungal infection, and protein content did not vary between leaves selected by females and unmined leaves, or between leaves with and without endophyte infections. We conclude that female Cameraria do not choose leaves within trees for oviposition on the basis of propensity for endophytic fungal infection or on phytochemical parameters that might indicate probability of future infections. At this spatial scale at least, fungal endophytes do not explain the highly aggregated distribution of Cameraria among leaves and associated costs in terms of increased larval mortality. Fungal endophytes may, nevertheless, affect leafminer dispersion and abundance at larger spatial scales, such as host plant populations or species. We did find, however, that the amount of mining activity on leaves is positively associated with increased colonization by fungal endophytes. We suggest that mining activity increases endophyte fungal infections by facilitating spore germination and hyphal penetration into the leaf or by altering leaf phytochemistry. The facilitation of endophyte colonization by leafmining activity coupled with the lack of predictability of endophyte infections based on leaf phytochemistry and almost 100% infectivity of all oak leaves during sporadic wet years may prevent female leafminers from discriminating leaves for oviposition on the basis of current or future levels of endophytes in leaves.     

Exploring the biocontrol potential of fungal endophytes from an Andean Colombian Paramo ecosystem

We studied the diversity and biocontrol potential of 100 fungal endophytes isolated from Espeletia spp., endemic plant species from the Paramo in the Andean mountain range. Our sample was genotypically highly diverse at all ITS similarity levels. The antagonistic properties of these isolates were tested against common crop pathogens in Colombia, including Pectobacterium carotovorum, Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas campestris, Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum, and Phytophthora infestans. All endophytic isolates were able to significantly inhibit the growth of at least one of the plant pathogens tested (P?<?0.05). Three main types of endophyte/pathogen interactions were observed. However, only those endophytes that produced an evident inhibition halo were further studied using their crude extracts to confirm that the inhibitory effect was due to the production of endophytic bioactive metabolites. From these experiments, nine promising isolates were selected for co-inoculation tests with R. solani in tomato plants. The isolates identified as Aureobasidium pullulans and Paraconiothyrium sporulosum not only protected the plants against this pathogen but also allowed them to exhibit similar growth and development as the uninoculated control. This work explores new alternatives for disease management without the application of chemical pesticides.     

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.     

Fungal endophytes associated with three South American Myrtae (Myrtaceae) exhibit preferences in the colonization at leaf level

Fungal endophytes associated with Myrtaceae from Brazil and Argentina were isolated at three levels of nesting: leaf, individual host trees, and site collection. The alternating logistic regression (ALR) was used to model the data because it offers a computationally convenient method for fitting regression structures involving large clusters. The objectives of this study were to determine: (i) whether the colonization pattern is influenced by environmental variables, (ii) if there is some leaf part they prefer to colonize; (iii) if there is some fungal endophyte aggregation between hierarchical levels; (iv) what the distance effect is on the fungal association. The environmental variables were statistically significant only for Xylaria, i.e., when the elevation and water precipitation increase and the temperature decreases, the odds ratio of finding another fungal endophyte of that genus previously found increases. Sordariomycetes, Xylariales, and Xylaria exhibited leaf fragment preference to petiole and tip. Fungal endophytes showed association within leaf. The horizontal transmission mode and the dispersal limitation may explain this association at the leaf level. Moreover, our results suggest that when a fungal endophyte infects a leaf or host tree individual, the odds ratio of dispersal inside them is greater.     

Response of Endophytic Bacterial Communities in Potato Plants to Infection with Erwinia carotovora subsp. atroseptica

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp. atroseptica, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the α, β, and γ subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group. Screening of the isolates for antagonistic activity against E. carotovora subsp. atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.