Fungal Endophyte Species Research Articles

Host associations and beta diversity of fungal endophyte communities in New Guinea rainforest trees.

Processes shaping the distribution of foliar fungal endophyte species remain poorly understood. Despite increasing evidence that these cryptic fungal symbionts of plants mediate interactions with pathogens and herbivores, there remain basic questions regarding the extent to which dispersal limitation and host specificity might shape fungal endophyte community composition in rainforests. To assess the relative importance of spatial pattern and host specificity, we isolated fungi from a sample of mapped trees in lowland Papua New Guinea. Sequences of the internal transcribed spacer (ITS) region were obtained for 2079 fungal endophytes from three sites and clustered into molecular operational taxonomic units (MOTUs) at 95% similarity. Multivariate analyses suggest that host affinity plays a significant role in structuring endophyte community composition whereas there was no evidence of endophyte spatial pattern at the scale of tens to hundreds of metres. Differences in endophyte communities between sampled trees were weakly correlated with variation in foliar traits but not with tree species relatedness. The dominance of relatively few generalist endophytes and the presence of a large number of rare MOTUs was a consistent observation at three sites separated by hundreds of kilometres and regional turnover was low. Host specificity appears to play a relatively weak but more important role than dispersal limitation in shaping the distribution of fungal endophyte communities in New Guinea forests. Our results suggest that in the absence of strong ecological gradients and host turnover, beta diversity of endophyte communities could be low in large areas of contiguous forest.

Impact of Domestication on the Endophytic Fungal Diversity Associated With Wild Zingiberaceae at Mount Halimun Salak National Park

Mount Halimun Salak National Park is one of the tropical forest remnants in Java island. The national park has been recognized with high diversity of wild Zingiberaceae. Of that Zingiberaceae, two species namely Alpinia malaccensis (AM) and Horstendia conica (HC), were domesticated as garden plants in the surrounding area of the forest for medicinal use. The impact of domestication on the fungal endophytes associated with these two species of Zingiberaceae is reported here. Fungal endophyte diversity in the wild and domesticated AM and HC was analyzed based on the culturable fungi. Identification of species level used morphological and molecular approaches of ITS rDNA sequence. This study determined 19 species of fungal endophytes, namely Arthrinium malaysianum, Aspergillus flavipes, As. sydowii, Chaetomium globosum, Cladosporium oxysporum, Cladosporium sp., Colletotrichum boninense-complex, Co. cliviae-complex, Co. gloeosporioides-complex, Diaporthe sp., D. anacardii, D. gardenia, Exophiala sp., E. lecanii-corni, Guignardia mangiferae, Ochroconis gallopava, Penicillium citrinum, Pyricularia costina, and unsporulated Sydowiellaceae. Among them, A. malaysianum, C. globosum, Co. cliviae-complex, D. gardenia, and unsporulated Sydowiellaceae were only found in domesticated plants, while some others were absent. Colletotrichum boninense-complex was commonly found in both wild and domesticated plants. Domestication activity affected the diversity of endophytic fungi of AM and HC.

Antagonistic activity of fungal endophyte filtrates against Gremmeniella abietina infections on Aleppo pine seedlings

Biological control agents (BCAs), and among them some species of fungal endophytes, are potential substitutes for chemical pesticides in the control of plant diseases due to their non-toxicity to human beings and their surrounding environment. One mode of action of fungal BCAs is through their bioactive, extracellular products, which can inhibit the growth of pathogens. In this study, the effect of fungal filtrates from four endophyte isolates (Trichoderma viride, Aureobasidium pullulans, Aureobasidium sp. and the unknown endophyte 20.1) on the advance of the pathogen Gremmeniella abietina on 2-year Pinus halepensis seedlings was evaluated. Both preventive and therapeutic treatments of the filtrates were studied by applying the filtrates either before or after the pathogen inoculation, respectively. Since G. abietina is a necrotrophic fungus, the length of the necrosis produced by the pathogen was used as response variable in our experiment. In order to explore the chemical composition of the fungal filtrates, a simple HPLC screening of UV-absorbing components was conducted. The results of the study showed that all fungal filtrates were able to reduce the advance of G. abietina when compared to the control seedlings, regardless of the time of inoculation and the treatment. Low-molecular weight phenolic compounds could be detected in some but not all filtrates, warranting further studies on the possible role of these compounds in fungal filtrates.

Diversity of Endophytic Fungi Isolated from Korean Ginseng Leaves

We investigated the diversity of the foliar endophytes of Korean ginseng. Endophytic fungi were isolated from healthy leaves of mountain-cultivated ginseng (MCG) and field-cultivated ginseng (FCG) at 4 sites in Chungbuk Province. A total of 24 species of fungal endophytes were identified using molecular approaches. Additionally, the diversity of these endophytic fungi was compared between MCG and FCG. The major isolated endophytes were Edenia gomezpompae and Gibberella moniliformis in the MCG and FCG samples, respectively. The results suggest that ginseng endophytes have different community structures in different environments, and this understanding may prove useful in ginseng cultivation.

Preliminary study of endophytic fungi in timothy (Phleum pratense) in Estonia

Timothy (<em>Phleum pratense </em>L.) is an important agricultural grass in Europe and North America, but there is little research into the occurrence and abundance of fungal endophyte species associated with this grass. The aim of this study was to identify fungal endophytes living within <em>P. </em>pratense and to determine if additional moisture applied during the growing season increases the diversity of endophytic fungi. We studied 58 isolates obtained from surface-sterilised blades of 60 <em>P. pratense </em>plants collected from Rõka Free Air Humidity Manipulation experimental plots (FAHM), Estonia. Morphological and molecular methods were used for isolate identification. As a result, 45 strains from 10 different taxa were identified, all belonging to Ascomycota. Five species were found to be new to <em>P. pratense</em>.

Comparative analysis of the biodiversity of fungal endophytes in insect-induced galls and surrounding foliar tissue

Insect-induced galls are abnormal plant growths that can provide food and shelter to their inhabitants, resulting in stressed plant tissue that may alter the conditions for the colonization or proliferation of endophytic fungi. We investigated the effect gall formation has on fungal endophyte communities and diversity. Using three closely-related gall-forming aphid species that specialize on poplars, we characterized fungal endophyte diversity in galls and surrounding petiole and leaf lamina tissue. A total of 516 fungal endophyte samples were isolated from 272 tissue samples (32 leaves, 31 petioles, and 209 galls), resulting in 23 distinct morphotypes. Despite sharing a common host plant and often forming spatially contiguous galls, the endophyte profiles within the galls of each aphid species were distinct, not only from the galls of the other species, but also from surrounding plant tissue. These results suggest that insect galls can affect the composition of fungal endophyte species in plant tissues, by altering either the colonization or proliferation of their endophytic mycobiota. Likewise, fungal endophytes may be important in the ecology and evolution of insect galls.

Molecular evidence for Neotyphodium fungal endophyte variation and specificity within host grass species

Host specificity of Neotyphodium species symbiotic with three grass species, Festuca arundinacea, Festuca pratensis and Lolium perenne, was studied based on comparisons of amplified fragment length polymorphisms (AFLP) between hosts and their corresponding endophytes. Endophytic fungi were isolated from 24 accessions of host plants. Neotyphodium identity was determined based on morphological characteristics observed in cultures and polymerase chain reaction analysis using specific primers. The results of AFLP data analysis revealed high genetic variation in plant and fungal endophyte species. Plant AFLP genotypes from different species clustered in three distinctive groups, congruent with species. A cluster analysis of AFLP data grouped endophytic isolates according to their host species and secondarily according to their host geographic distribution. The result of the AMOVA on AFLP data accounted for a large and significant proportion of genetic variation due to differences among plant and endophyte species. Phylogenetic groups of isolates corresponded to their respective host genotypes based on maximum parsimony phylograms. Comparisons of the two phylograms illustrated a significant congruence between nodes and branches of host and endophyte clades. These results strongly suggest host specificity of Neotyphodium fungal endophytes with their geographically distant host grasses within each species.

Age-dependent Distribution of Fungal Endophytes in Panax ginseng Roots Cultivated in Korea.

Fungal endophytes were isolated from 1-, 2-, 3-, and 4-year-old ginseng roots (Panax ginseng Meyer) cultivated in Korea. The isolated fungal endophytes were identified based on sequence analysis of the internal transcribed spacer and morphological characterization by microscopic observations. A total of 81 fungal endophytes were isolated from 24 ginseng roots. Fungal endophytes were classified into 9 different fungal species and 2 unknown species. Ginseng roots that were 1-, 2-, 3-, and 4-years old were colonized by 2, 6, 8, and 5 species of fungal endophytes, respectively. While Phoma radicina was the most frequent fungal endophyte in 2-, 3-, and 4-year-old ginseng roots, Fusarium solani was the dominant endophyte in 1-year-old ginseng roots. The colonization frequencies (CF) varied with the host age. The CF were 12%, 40%, 31%, and 40% for 1-, 2-, 3-, and 4-year-old ginseng roots, respectively. We found a variety of fungal endophytes that were distributed depending on the age of ginseng plants.

Species and organ specificity of fungal endophytes in herbaceous grassland plants

Summary1. aFoliar endophytic fungi in herbaceous plants are known to be diverse, yet studies characterizing the fungal communities in roots and shoots of plants across time are absent. These fungi are supposedly ubiquitous in nature, infecting plants through airborne spores. As many foliar endophytes can also exist in soil, we hypothesized that there would be a strong similarity between root and shoot endophytes in any given plant species and that differences between plant species would be greater than those between organs within a species.2. Leaf and root fungal endophyte communities were assessed in field‐collected plants of three co‐occurring grassland forbs (Cirsium arvense, Plantago lanceolata and Rumex acetosa) in two contrasting seasons (winter and summer). The former two species are mycorrhizal, whilst the latter species is not so.3. The highest fungal species richness was recorded from P. lanceolata with R. acetosa hosting the least endophyte diversity. Endophyte communities were more diverse in root than leaf tissues and in summer than in winter. Similarity of endophyte communities between different host plant species and organs within an individual host was very low. Negative correlations were found between mycorrhizal colonization and endophyte presence in the roots of C. arvense and P. lanceolata, suggesting some degree of antagonism or competition between the fungi. Consistent positive associations were found between the number of endophyte species in roots and shoots of plants.4. Synthesis. In contrast to previous studies, the results show that many endophytes do not occur ubiquitously, but instead exhibit both plant and tissue specificity. There is a strong seasonal change in endophyte communities, but the differences between roots and shoots at any one time can be just as large. This dissimilarity suggests a lack of systemic growth by the fungi from one tissue to another. Mycorrhizas may interact negatively with other root endophytes, indicating that the latter should not be ignored in future mycorrhizal studies. We should begin to think of individual plants as ecosystems of interacting microbes, whose community is structured by plant genetics and environmental conditions, coupled with interactions between the microbes themselves.

Kit for detection of fungal endophytes of grasses yields inconsistent results

Summary1. The Agrinostics field immunoblot assay is a colour‐imprint assay originally developed for routinely and rapid detection of mycotoxin‐producing fungi Neotyphodium spp./Epichloë spp. from samples of pasture, turf, cereals and seeds of agricultural grasses.2. In addition the Agrinostics immunoblot assay has been used and recommended for exploring the presence and ecological role of Neotyphodium and the closely related Epichloë in several cool season grasses.3. Here we demonstrate that this method is not limited to detection of only these two species, as several other fungal endophyte genera or species also yielded positive results when mycelia were loaded onto cards. Moreover, other closely related endophyte genera or species were found to yield negative result. Thus, the assay is neither specific to isolates of Neotyphodium/Epichloë nor is it universal for fungal endophyte detection.4. Our study underscore the fact that the Agrinostics field immunoblot assay should not be used on its own for field research into the broader ecological significance of Neotyphodium/Epichloë.5. We suggest that the assay may be best suited for use in agricultural fields or in controlled greenhouse experiments for hyphal load estimates, and should always be accompanied by other methods (e.g. microscopic visualization, DNA‐sequencing) to confirm infection frequencies and certify that the endophyte present actually is Neotyphodium/Epichloë.

Exploring the evolutionary ecology of fungal endophytes in agricultural systems: using functional traits to reveal mechanisms in community processes.

All plants, including crop species, harbor a community of fungal endophyte species, yet we know little about the biotic factors that are important in endophyte community assembly. We suggest that the most direct route to understanding the mechanisms underlying community assembly is through the study of functional trait variation in the host and its fungal consortium. We review studies on crop endophytes that investigate plant and fungal traits likely to be important in endophyte community processes. We focus on approaches that could speed detection of general trends in endophyte community assembly: (i) use of the ‘assembly rules’ concept to identify specific mechanisms that influence endophyte community dynamics, (ii) measurement of functional trait variation in plants and fungi to better understand endophyte community processes and plant–fungal interactions, and (iii) investigation of microbe–microbe interactions, and fungal traits that mediate them. This approach is well suited for research in agricultural systems, where pair-wise host–fungus interactions and mechanisms of fungal–fungal competition have frequently been described. Areas for consideration include the possibility that human manipulation of crop phenotype and deployment of fungal biocontrol species can significantly influence endophyte community assembly. Evaluation of endophyte assembly rules may help to fine-tune crop management strategies.

Direct classification of related species of fungal endophytes (Epichloëspp.) using visible and near-infrared spectroscopy and multivariate analysis

The aim of this work was to investigate the potential of visible and near-infrared (Vis-NIR) reflectance spectroscopy for the classification of three morphologically similar species of fungal endophytes of grasses. Vis-NIR spectra (400-2498 nm) from 34 isolates of Epichloë sylvatica, 32 of Epichloë typhina and 38 of Epichloë festucae were recorded directly from fresh mycelium growing in potato dextrose agar plates. Multivariate procedures applied to the spectral data were discriminant modified partial least squares regression, soft independent modelling of class analogy and discriminant partial least squares regressions (PLS1, PLS2). Several types of data pretreatments were tested to develop the classification models. The best predictive models were achieved with PLS2 analysis; with this method, 90% of E. typhina and 100% of E. festucae and E. sylvatica external validation samples were successfully classified. These results show the potential of Vis-NIR spectroscopy combined with multivariate analysis as a rapid method for classifying morphologically similar species of filamentous fungi.

Molecular detection of Neonectria galligena (syn. Nectria galligena)

A pair of primers specific for Neonectria galligena were designed from comparisons of the internal transcribed spacer (ITS) regions 1 and 2 of 32 isolates of diverse origins with sequences from 7 other nectriaceous species: ‘Nectria’ ditissima, N. coccinea, N. coccinea var. faginata, N. punicea, N. fuckeliana, N. cinnabarina , and N. radicicola. Comparisons were also made with sequences from several commonly encountered fungal endophyte species of apple wood, in particular Fusarium lateritium. Under stringent PCR conditions, primers Ch1 and Ch2 amplify a 412 bp fragment from N. galligena DNA nectriaceous but not from other nectriaceous species, fungal apple endophyte species, rosaceous, or beech host tissues. The identity of PCR products from total DNA extracts from woody tissues was confirmed by Southern hybridization. A 500 bp heterologous internal standard incorporating identical primer recognition sites was generated for use as an internal positive control for individual PCR assays to identify false negatives due to the presence of high levels of co-precipitated PCR inhibitory compounds. Further adaptation of this approach allowed for semi-quantitative competitive PCR enabling the determination of target fungal levels in infected lignified tissues.

DO FUNGAL ENDOPHYTES RESULT IN SELECTION FOR LEAFMINER OVIPOSITIONAL PREFERENCE?

Microorganisms can alter the direction and magnitude of plant–herbivore interactions. However, how they affect the susceptibility of the host plant and their impact on the herbivore may vary depending on the scale of the interaction such as among locations, individual plants, and parts of the plant. Positive preference–performance relationships for insects in general, and for sedentary insects such as leafminers in particular, would predict that there would be negative selection pressure against females ovipositing in low-performance sites. Thus, where the presence of microorganisms causes reduced performance, selection would favor oviposition in sites where the microbes are absent. However, this assumes that ovipositing females can detect the microbes and respond by avoidance, or that females can use cues that predict the future presence of microbes and use these predictors to respond by avoidance. We used the Cameraria sp. leafminer–Quercus emoryi system and the associated fungal endophyte species that ...

Do Fungal Endophytes Result in Selection for Leafminer Ovipositional Preference?

Microorganisms can alter the direction and magnitude of plant–herbivore interactions. However, how they affect the susceptibility of the host plant and their impact on the herbivore may vary depending on the scale of the interaction such as among locations, individual plants, and parts of the plant. Positive preference–performance relationships for insects in general, and for sedentary insects such as leafminers in particular, would predict that there would be negative selection pressure against females ovipositing in low-performance sites. Thus, where the presence of microorganisms causes reduced performance, selection would favor oviposition in sites where the microbes are absent. However, this assumes that ovipositing females can detect the microbes and respond by avoidance, or that females can use cues that predict the future presence of microbes and use these predictors to respond by avoidance. We used the Cameraria sp. leafminer–Quercus emoryi system and the associated fungal endophyte species that share the leaves of the host to test the hypothesis that leafminer ovipositional preference and therefore distribution are negatively associated with endophyte distribution at scales where endophytes mediate insect antagonism and where endophyte distribution is predictable. We censused fungal endophyte distribution and abundance on >20 trees over three growing seasons at the following scales: among trees, among positions within trees, among leaves, and between and within seasons. In addition, we censused leafminer distribution and abundance and used data on leafminer distributions at different spatial scales collected over the past decade from other studies in this system to determine how they are associated with endophytes. We found that leafminers and fungal endophytes were negatively associated at two spatial scales where endophytes have been shown to affect leafminer performance negatively and where endophyte distribution and abundance were predictable. Endophytes and leafminers were not associated at one spatial scale where endophytes do not affect leafminers, and distribution and abundance was not predictable. Leafminer growth and endophyte infection were positively associated over time as both increase over the season. The results demonstrate that leafminer ovipositional preference may have been selected by avoidance of fungal endophytes and associated endophyte-mediated antagonism, but that avoidance is reliant on predictable patterns of infection.

Fungal Endophytes in Oak Trees: Long-Term Patterns of Abundance and Associations with Leafminers

Endophytic fungi inhabiting foliage of woody plants are diverse and abundant, yet little is known of their temporal and spatial variation or their interactions with more commonly studied invertebrate and vertebrate macroherbivores. We studied seasonal and yearly variation in endophyte infections in five Emory oak trees over a 4-yr period from 1989 to 1993. In the 1992–1993 growing season, we documented seasonal patterns of abundance of the three most common fungal endophyte species, an ascomycete, Ophiognomonia cryptica, and two coelomycete species, Plectophomella sp. and Asteromella sp. To determine the association of fungal endophytes with macroherbivores, we compared infection frequencies of endophytes in unmined leaves and leaves harboring living and dead larval leafminers of Cameraria sp. nov., the dominant macroherbivore on Emory oak, over the 4-yr period. In the 1992–1993 growing season, we monitored survival and mortality of leafmining larvae on the five study trees. We then determined the association of total infections and infections by individual endophyte species with seasonal survival and sources of mortality of leafminers. Infection levels of endophytes varied over the 4-yr period, but seasonal patterns within years were consistent. The three dominant species of endophytes varied differently in frequencies over the growing season. Leafminers were positively associated with endophytic infections in all years. In two of the four years, leaves with living leafmining larvae had greater total infection frequencies than unmined leaves. In one of these years, infection frequencies were greater in leaves with dead larvae than in those with living larvae, suggesting that endophytes may increase mortality of leafminers. However, when infection levels of individual species were compared among leaf types, only levels of Asteromella were greater in mined leaves relative to unmined ones. This difference was explained largely by higher infection levels in leaves with living larvae than in leaves with dead larvae. Furthermore, seasonal changes in endophyte levels varied independently from seasonal changes in leafminer mortality from unexplained causes or natural enemies. We conclude that leafmining activity is associated with higher endophyte infections, but increased infections are not associated with increased leafminer mortality. Higher infections on mined leaves are likely caused by leafmining damage that enhances fungal colonization and penetration into oak leaves. Although endophytes of Emory oak are diverse and abundant and their presence is associated with leafmining activity, we do not find strong correlative evidence that endophytes interact mutualistically with Emory oak by increasing resistance to a dominant herbivore, the leafminer Cameraria. A companion paper (Faeth and Hammon 1997) corroborates experimentally the lack of antagonistic interaction between endophytes and the leafminer.

FUNGAL ENDOPHYTES IN OAK TREES: LONG-TERM PATTERNS OF ABUNDANCE AND ASSOCIATIONS WITH LEAFMINERS

Endophytic fungi inhabiting foliage of woody plants are diverse and abundant, yet little is known of their temporal and spatial variation or their interactions with more commonly studied invertebrate and vertebrate macroherbivores. We studied seasonal and yearly variation in endophyte infections in five Emory oak trees over a 4-yr period from 1989 to 1993. In the 1992–1993 growing season, we documented seasonal patterns of abundance of the three most common fungal endophyte species, an ascomycete, Ophiognomonia cryptica, and two coelomycete species, Plectophomella sp. and Asteromella sp. To determine the association of fungal endophytes with macroherbivores, we compared infection frequencies of endophytes in unmined leaves and leaves harboring living and dead larval leafminers of Cameraria sp. nov., the dominant macroherbivore on Emory oak, over the 4-yr period. In the 1992–1993 growing season, we monitored survival and mortality of leafmining larvae on the five study trees. We then determined the association of total infections and infections by individual endophyte species with seasonal survival and sources of mortality of leafminers. Infection levels of endophytes varied over the 4-yr period, but seasonal patterns within years were consistent. The three dominant species of endophytes varied differently in frequencies over the growing season. Leafminers were positively associated with endophytic infections in all years. In two of the four years, leaves with living leafmining larvae had greater total infection frequencies than unmined leaves. In one of these years, infection frequencies were greater in leaves with dead larvae than in those with living larvae, suggesting that endophytes may increase mortality of leafminers. However, when infection levels of individual species were compared among leaf types, only levels of Asteromella were greater in mined leaves relative to unmined ones. This difference was explained largely by higher infection levels in leaves with living larvae than in leaves with dead larvae. Furthermore, seasonal changes in endophyte levels varied independently from seasonal changes in leafminer mortality from unexplained causes or natural enemies. We conclude that leafmining activity is associated with higher endophyte infections, but increased infections are not associated with increased leafminer mortality. Higher infections on mined leaves are likely caused by leafmining damage that enhances fungal colonization and penetration into oak leaves. Although endophytes of Emory oak are diverse and abundant and their presence is associated with leafmining activity, we do not find strong correlative evidence that endophytes interact mutualistically with Emory oak by increasing resistance to a dominant herbivore, the leafminer Cameraria. A companion paper (Faeth and Hammon 1997) corroborates experimentally the lack of antagonistic interaction between endophytes and the leafminer.

Importance of Acremonium endophytes in turf-grass breeding and management

The development and use of genetically improved turf-grasses containing useful endophytes is of great value in efforts to enhance the environment, reduce maintenance cost, and conserve and improve precious soil and water resources. Acremonium endophyte-infected (E+) turg-grasses have frequently shown dramatic enhancement of resistance to many foliage-feeding insect pests. Modest improvements in resistance to root-feeding white grubs and some nematodes have been reported. Improvements in stress tolerance and modifications in water relations have been observed under certain conditions. Acremonium endophytes cause modifications to physiological mechanisms of adaptation of the host that result in superior performance of some turf-grass genotypes during moisture deficits. An understanding of the role of endophytes in modifying host plant performance makes turf-grass breeding and evaluation much more efficient. During 1990, an estimated 6000 tonnes of seed of elite, E+ containing perennial ryegrasses, Lolium perenne L., was produced and utilized throughout the world. Endophyte-enhanced ryegrass cultivars include ‘Advent’, ‘Assure’, ‘Dandy’, ‘Dasher II’, ‘Gettysburg’, ‘Pinnacle’, ‘Repell’, ‘SR 4200’, ‘Seville’, and ‘Sherwood’. Acremonium endophytes have been incorporated into ‘Reliant’ and ‘SR 3000’ hard fescue, Festuca longifolia Thuill, ‘SR 5000’ and ‘Jamestown II’ Chewings fescue, Festuca rubra L. subsp. commutata Gaud., as well as elite germplasm of these species. Lower-growing, turf-type tall fescues, Festuca arundinacea Schreb., are being developed with high infection levels of useful Acremonium endophytes. Efforts are being made to find or develop and use desirable endophytes in Kentucky bluegrass, Poa pratensis L., strong creeping red fescue, F. rubra L. subsp. rubra, blue fescue, Festuca glauca Lam., and various bentgrasses, Agrostis spp. The discovery and/or development of superior genetic strains of Acremonium endophytes will make endophyte-enhanced turf-grass performance of even greater significance. There is also the potential to discover, genetically modify, and use other species of fungal and bacterial endophytes with the ability to improve turf-grass performance.