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PubMed1998

Endophytic fungi in indigenous Australasian grasses associated with toxicity to livestock.

Grazing of Echinopogon spp. by livestock in Australia has caused symptoms similar to those of perennial ryegrass staggers. We observed an endophytic fungus in the intercellular spaces of the leaves and seeds of New Zealand and Australian specimens of Echinopogon ovatus. Culture of surface-sterilized seeds from New Zealand specimens yielded a slow-growing fungus. An examination in which immunoblotting and an enzyme-linked immunosorbent assay (ELISA) were used indicated that E. ovatus plants from Australia and New Zealand were infected with fungi serologically related to Neotyphodium lolii (the endophyte of perennial ryegrass) and other Epichloe and Neotyphodium spp. endophytic in pooid grasses. No lolitrems (the indole-diterpenoids implicated as the causative agents of perennial ryegrass staggers), peramine analogs, or ergot alkaloids were detected in the infected specimens by high-performance liquid chromatography or ELISA. However, in endophyte-infected E. ovatus plants from New Zealand, analogs of the indole-diterpenoid paxilline (thought to be a biosynthetic precursor of the lolitrems and related tremorgens) were detected by ELISA, and N-formylloline was detected by gas chromatography. Endophyte-free specimens of New Zealand E. ovatus did not contain detectable paxilline analogs or lolines and were more palatable than infected specimens to adults of the pasture pest Listronotus bonariensis (Argentine stem weevil). Hyphae similar to those of the E. ovatus endophyte were also found in herbarium specimens of Echinopogon nutans var. major, Echinopogon intermedius, Echinopogon caespitosus, and Echinopogon cheeli. This appears to be the first time that an endophytic Neotyphodium species has been identified in grasses endemic to New Zealand or Australia.

Acremonium↗

PubMed2002

Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants.

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log(10) CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log(10) CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log(10) CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log(10) CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.

Bacteria↗

PubMed1975

Demonstration of the isolation of non-infective Alnus crispa var. mollis Fern, nodule endophyte by morphological immunolabelling and whole cell composition studies.

Two filamentous, branched, and septate actinomycetes were isolated from field-collected and from axenic in vitro produced root nodules of Alnus crispa var. mollis Fern. host plant. After their transfer to a chemically defined medium, these nodule isolates could not be distinguished from each other on the basis of morphology, cultural reactions, and whole cell composition and were considered to be the same species. They were morphologically similar to the root nodule endophyte, but were incapable of nodulating aseptic host plants growing in a nitrogen-deficient substrate. Whole cells of the nodule isolates were used for the production of rabbit antibodies. The resulting specific antiisolate antibodies were conjugated with fluorescein isothiocyanate and used in staining tests of the nodule endophyte. The immunofluorescence reactions demonstrated the homology of the nodule isolates with the nodule endophyte. After pectinase degradation of the endophyte capsule, the indirect immunoferritin method corroborated the fluorescent anti-body (FA) staining reactions. There was no antigenic relationship between the nodule isolates and 13 known strains of actinomycetes as determined by the FA techique. Fluorescent antibody reactions of adsorbed conjugates suggested that endophytes of both Alnus crispa var. mollis Fern. and Alnus rugosa (DuRoi) Spreng. root nodules belong to a common serotype. The LL and mesoisomers of diaminopimelic acid were present in similar proportions in the nodule endophyte and in the nodule isolates. Glucose, mannose, and an unknown sugar were the predominant whole cell sugars in the nodule isolates, although trace amounts of arabinose and rhamnose were also displayed. The unknown sugar found in the nodule isolates was also present in trace amounts in the endophyte-suspension hydrolysate.

Actinomycetales↗

PubMed2001

An endophyte of the tropical forage grass Brachiaria brizantha: isolating, identifying, and characterizing the fungus, and determining its antimycotic properties.

Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C4 grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.

Acremonium↗

PubMed2004

Endophytic fungal diversity of 2 sand dune wild legumes from the southwest coast of India.

Endophytic fungi of 3 age classes (seeds, seedlings, and mature plants) and 5 tissue classes (cotyledons, seed coats, roots, stems, and leaves) of coastal sand dune legumes Canavalia cathartica and Canavalia maritima were assessed by plating surface-sterilized segments on malt extract agar. Forty-six fungal taxa comprising 6 ascomycetes, 33 mitosporic fungi, 2 zygomycetes, and 5 sterile morphospecies were recovered. There was no significant difference in the colonization frequency of endophytes between plant species (p = 0.4098, Student's t test). Among the age classes, endophytic fungi colonized over 90% of seedlings and mature plants. Similarly, among tissue classes, endophytic fungi colonized over 90% of root, stem, and leaf segments. Diversity and richness of endophytic fungi were higher in C. cathartica than in C. maritima. Rarefaction curves revealed a "higher expected number of species" in mature plants of C. cathartica and seedlings of C. maritima, whereas it was highest in leaves of both plant species. The most dominant endophyte, Chaetomium globosum, colonized over 50% of the root, stem, and leaf segments of C. maritima and over 50% of the root segments of C. cathartica. The colonization frequency of C. globosum was found to be 5%-12.5% in seeds and increased up to 40%-64.4% in seedlings or mature plants. Halosarpheia sp. was the only marine fungus recovered among the endophytes.

Canavalia↗

PubMed1995

Toxic endophyte-infected tall fescue and range grasses: historic perspectives.

A historic profile of endophyte-induced tall fescue toxicosis is presented. A chronology of events is presented, beginning with the importance of finding Balansia-infected grasses in a tall fescue pasture in north central Georgia. This initial finding was followed by the discovery that another related endophyte was present in tall fescue and other major forage grasses. This species of endophyte was identified as Acremonium coenophialum. After this report was the important discovery that cattle performance was poor on Acremonium-infected tall fescue. Thus, this endophyte and its presence in tall fescue was implicated as the cause of tall fescue toxicosis and fescue foot. It was later established that this endophyte also produced ergot alkaloids. The related endophyte of perennial ryegrass, A. lolii, was subsequently shown to be responsible for the ryegrass staggers syndrome. Several other species of Acremonium have been associated with other important forage and turf grasses. Finally, important papers leading to the revelations that endophytic fungi and their grass hosts are ecologically significant and that most should be considered mutualistic symbioses are reviewed. Symbiotic grasses have enhanced physiological and morphological characteristics that offer biotechnological exploitations on one hand, but on the other solutions to the toxicity of tall fescue are difficult because grasses free of their fungal partner are generally ecological failures.

Acremonium↗

PubMed2003

Use of nonergot alkaloid-producing endophytes for alleviating tall fescue toxicosis in sheep.

Nonergot alkaloid-producing endophytes from New Zealand were inserted into tall fescue (Festuca arundinacea) cultivars in an attempt to address the problem of fescue toxicosis in grazing sheep. A 3-yr grazing study was conducted to determine lamb performance and to evaluate toxicosis in lambs grazing nonergot alkaloid-producing endophyte-infected (AR542 or AR502), endophyte-free (E-), or wild-type toxic endophyte-infected (E+) Jesup tall fescue or nonergot alkaloid-producing endophyte-infected (AR542) Georgia-5 tall fescue. Replicated 0.11-ha tall fescue paddocks were established at the central Georgia Branch Station during September 1997 and stocked with lambs from spring 1998 through autumn 2000. Mean ergot alkaloid concentrations were higher (P < 0.01) in E+ forage than in AR542, AR502, and E- tall fescue, and ergot alkaloid concentrations in E- plants and plants infected with AR542 and AR502 were low. Forage availability did not differ (P = 0.92) across treatments during autumn and was higher (P < 0.05) in Georgia-5 AR542 than in Jesup AR502 and E+ pastures. Initial serum prolactin (PRL) concentrations did not differ (P = 0.58) across treatments during autumn, but were higher on Jesup AR542 than E+ during spring. Post-treatment serum PRL concentrations were depressed (P < 0.01) on E+ compared with AR542, AR502, and E- in both spring and autumn. Signs of heat stress were observed in E+ lambs during periods of high ambient temperatures. Mean post-treatment rectal temperature and mean stocking rate exhibited treatment x year interactions (P < 0.05). Lamb ADG was higher (P < 0.05) on AR542, AR502, and E- than on E+ tall fescue. Similarly, gain/hectare was higher (P < 0.015) on AR542, AR502, and E- than on E+. Tall fescue pastures containing AR542 and AR502 endophytes yielded lamb performance that did not differ from that on E- tall fescue and which was superior to performance on E+ tall fescue. Depressed PRL concentrations and elevated rectal temperatures as indicators of toxicosis were evident only in lambs grazing E+ tall fescue, suggesting that nonergot alkaloid-producing endophyte-infected tall fescue is a viable alternative for alleviating tall fescue toxicosis.

Animal Feed↗

PubMed2005

Fungal endophytes in five medicinal plant species from Kudremukh Range, Western Ghats of India.

Eighteen species of endophytic fungi were isolated from bark, stem and leaf segments of five medicinal plant species growing within the Kudremukh range in the Western Ghats of India. The dominant endophytic fungal species isolated from these plant species were Curvularia clavata, C. lunata, C. pallescens and Fusarium oxysporum. The highest species richness as well as frequency of colonization of endophytic fungi was found in the leaf segments, rather than the stem and bark segments, of the host plant species. The greatest number of endophytic fungal species were found within Callicarpa tomentosa (11 species), whereas Lobelia nicotinifolia harbored the lowest number of fungal endophytes (5 species). This study provides evidence that fungal endophytes are host and tissue specific.

Ascomycota↗

PubMed2004

Are endophyte-mediated effects on herbivores conditional on soil nutrients?

Neotyphodium endophytes are assumed to have mutualistic relationship with their grass hosts, mainly resulting from mycotoxin production increasing plant resistance to herbivores by the fungus that subsists on the plant. To study importance of often ignored environmental effects on these associations, we performed a greenhouse experiment to examine the significance of endophyte infection and nutrient availability for bird-cherry aphid ( Rhopalosiphum padi) performance on meadow fescue ( Lolium pratense). Naturally endophyte-infected (E+), uninfected (E-), or manipulatively endophyte-free (ME-) half-sib families of meadow fescue were grown on two soil nutrient levels. Endophyte infection reduced aphid performance in general. However, to our knowledge, this is the first study to demonstrate experimentally that herbivore performance decreases on E+ host plants with increasing availability of nutrients in soils. Potential improvement in herbivore performance in high nutrient soils and decreased plant performance in low nutrient soils in ME- plants, compared to E- and E+ plants, suggests that loss of endophyte infection after long coevolutionary relationship may be critical to plant fitness.

Animals↗

PubMed2006

Peramine and other fungal alkaloids are exuded in the guttation fluid of endophyte-infected grasses.

Many grasses live in association with asymptomatic fungi (Neotyphodium spp. endophytes), which grow in the intercellular spaces of the grass. These endophytes produce a range of alkaloids that protect the grass against grazing by mammals and insects. One of these alkaloids is an unusual pyrrolopyrazine, peramine. Peramine appears to be continuously produced by the endophyte, but does not progressively accumulate. No mechanism for the removal of peramine by its further metabolism or any other process has been reported. Our aim was to detect peramine or peramine metabolites in plant fluids to determine if peramine is mobilized, metabolized or excreted by the plant. We also wanted to determine if other fungal metabolites are mobilized by the plant, as has been proposed for the loline alkaloids. We developed a highly sensitive method for the analysis of peramine, using a linear ion trap mass spectrometer. We studied the fragmentation pathway of peramine using ESI MSn and ESI FTICRMS. Based on these results we developed a single reaction monitoring method using the fragmentation of the guanidinium moiety. Cut leaf fluid and guttation fluid of different grass endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloë sp.) were analysed. Peramine was detected in the cut leaf fluid of all grass-endophyte associations, but not in the guttation fluid of all associations. In some associations we also detected lolines and ergot peptide alkaloids. This is the first report showing the mobilization of fungal alkaloids into plant fluids by the host plant in grass-endophyte associations.

Alkaloids↗

PubMed2006

Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages.

Although associated with all plants, fungal endophytes (microfungi that live within healthy plant tissues) represent an unknown proportion of fungal diversity. While there is a growing appreciation of their ecological importance and human uses, little is known about their host specificity, geographic structure, or phylogenetic relationships. We surveyed endophytic Ascomycota from healthy photosynthetic tissues of three plant species (Huperzia selago, Picea mariana, and Dryas integrifolia, representing lycophytes, conifers, and angiosperms, respectively) in northern and southern boreal forest (Québec, Canada) and arctic tundra (Nunavut, Canada). Endophytes were recovered from all plant species surveyed, and were present in <1-41% of 2 mm2 tissue segments examined per host species. Sequence data from the nuclear ribosomal internal transcribed spacer region (ITS) were obtained for 280 of 558 isolates. Species-accumulation curves based on ITS genotypes remained non-asymptotic, and bootstrap analyses indicated that a large number of genotypes remain to be found. The majority of genotypes were recovered from only a single host species, and only 6% of genotypes were shared between boreal and arctic communities. Two independent Bayesian analyses and a neighbor-joining bootstrapping analysis of combined data from the nuclear large and small ribosomal subunits (LSUrDNA, SSUrDNA; 2.4 kb) showed that boreal and arctic endophytes represent Dothideomycetes, Sordariomycetes, Chaetothyriomycetidae, Leotiomycetes, and Pezizomycetes. Many well-supported phylotypes contained only endophytes despite exhaustive sampling of available sequences of Ascomycota. Together, these data demonstrate greater than expected diversity of endophytes at high-latitude sites and provide a framework for assessing the evolution of these poorly known but ubiquitous symbionts of living plants.

Ascomycota↗

PubMed2001

Effects of elevated ultraviolet radiation and endophytic fungi on plant growth and insect feeding in Lolium perenne, Festuca rubra, F. arundinacea and F. pratensis.

Plants of perennial ryegrass (Lolium perenne L.), red fescue (Festuca rubra L.), tall fescue (F. arundinacea Schreb.) and meadow fescue (F. pratensis Huds) were exposed at an outdoor facility located in Edinburgh, UK to modulated levels of UV-B radiation (280-315 nm) using banks of cellulose diacetate filtered UV-B fluorescent lamps that also produce UV-A radiation (315-400 nm). The plants were derived from a single clone of each species and were grown both with and without colonization by naturally-occurring fungal endophytes. The UV-B treatment was a 30% elevation above the ambient erythemally-weighted level of UV-B during July to October. Growth of treated plants was compared with plants grown under elevated UV-A radiation alone produced by banks of polyester filtered lamps and with plants grown at ambient levels of solar radiation under banks of unenergized lamps. At the end of the treatment period, sample leaves were collected for feeding trials with the desert locust Schistocerca gregaria (Forsk). The UV-B treatment produced no effects on the aboveground biomass of any of the four grasses. The UV-B treatment and the UV-A control exposure both increased plant height and the number of daughter plants formed by rhizome growth in F. rubra. There were significant effects of endophyte presence on the total fresh and dry weights of F. arundinacea and F. rubra, on fresh weight only in F. pratensis, and on the fresh and dry weights of inflorescence in F. arundinacea and L. perenne. There were no effects of UV treatments on the absolute amounts of leaf consumed or on the feeding preferences of locusts for leaves with or without endophyte in three species: F. rubra, F. arundinacea and L. perenne. In F. pratensis there was no effect of UV treatment on the weight of leaves consumed but a significant UV x endophyte interaction caused by a marked change in feeding preference between leaves with and without endophyte that differed between the UV-B treatment and UV-A control exposures. The alkaloid compounds known as lolines were analysed in leaves of F. pratensis and were only found in plants grown with endophyte. However, there was no significant relationship between total loline content and insect feeding preference. These effects illustrate the potential complexities of species interactions under increasing levels of UV-B. The experiment also demonstrates the importance of appropriate controls in UV lamp supplementation experiments for interpretation of both plant growth and insect feeding effects.

Animals↗

PubMed2003

Some endophytic fungi reduce the density of pustules of Puccinia recondita f. sp. tritici in wheat.

The interaction between Puccinia recondita f. sp. tritici (now widely referred to as P. triticina) and endophytic fungi in wheat was examined in laboratory experiments to determine whether the presence of fungal endophytes suppresses leaf rust disease caused by this fungus. Endophytes and cell-free washings from culture plates of the endophytes reduced the density and size of pustules in a susceptible cultivar when inoculated 3, 7 and 14 d prior to the pathogen. Disease at 12 d was reduced when the endophytes were inoculated simultaneously up to 50 mm from the fungus. Interactions between endophytes and this Puccinia are most probably mediated by defence mechanisms induced in the host plant.

Ascomycota↗

PubMed2002

Decline in diversity and abundance of endophytic fungi in twigs of Fagus sylvatica L. after experimental long-term exposure to sodium dodecylbenzene sulphonate (SDBS) aerosol.

Sodium dodecylbenzene sulphonate (SDBS) is an anionic synthetic detergent found in polluted sea aerosol and is known for its harmful effects on leaf surface ultrastructure on conifers and broadleaved trees. Four-year-old saplings of European beech were sprayed weekly for three consecutive growing seasons with either a 50 mg l(-1) solution of SDBS in deionized water or with pure deionized water (control). Two- to three- year-old twigs were collected from SDBS-treated and control plants during the growing season one year after the last treatment to isolate endophytic fungi. The frequency of colonization by endophytic fungi was significantly lower on SDBS-treated plants (63.8%) than on control plants (85.4%). Multiple colonization of twigs occurred more frequently and diversity of endophyte species was higher in control plants than in SDBS-treated plants. Thirty-six fungal species were isolated from 360 twigs. Cladosporium cladosporioides, Coryneum compactum, Phialocephala dimorphospora, and a species each of Mycosphaerella and Phomopsis were the most abundant endophytes with frequencies of colonization of more than 5%. The abundance of the Phomopsis species proved to be significantly reduced by the SDBS treatment. Within the limits of the indoor experimental conditions, the obtained results suggest that long-term exposure of aerial parts of beech to SDBS can affect the amount and composition of endophytic fungal communities of lignified twigs. Degradation of the leaf epicuticular wax layer and changes of the assimilation capacity and leaf water content (transpiration) of the crowns are presumed to be responsible for the reduction of endophytic fungi detected in twigs of SDBS-treated plants.

Aerosols↗

PubMed2003

Mutualistic asexual endophytes in a native grass are usually parasitic.

Asexual systemic fungi that live symbiotically within grasses are viewed as strong mutualists on the basis of theory and empirical studies of introduced agronomic grasses. Evolutionary theory predicts that microbial symbionts that lose sexuality and rely on propagules of their hosts for transmission should evolve to benefit their hosts. Fungal endophytes of some cultivated turf and pasture grasses are well known for increasing plant performance and competitive abilities, especially under stress, and increasing resistance to herbivores, pathogens, and root-feeders by virtue of fungal alkaloids. The assumption of mutualism, however, has rarely been tested in native grasses, which often harbor high but variable frequencies of systemic asexual endophytes. We tested the effect of Neotyphodium infections for the native grass Arizona fescue in a 3-yr field experiment. We strictly controlled host genotype and manipulated soil moisture and nutrients. Infection generally decreased host growth in terms of plant volume, number of tillers, and dry mass of shoots and roots. Infected plants also showed decreased reproduction in terms of number and mass of seeds, and the seeds produced by infected plants had lower germination success than plants without their endophytes, suggesting that the negative effects of the symbiont are transferred to the next generation. Plant genotype strongly influenced host's growth and reproduction and interacted with the presence of the endophyte, but the interaction was usually in the direction of negative effects. Our results challenge the notion that systemic asexual endophytes must be plant mutualists for infections to persist in nature. We propose other hypotheses to explain the variable but usually high endophyte frequencies in natural populations of grasses.

Fungi↗

PubMed2005

Seasonal variations in location and population structure of endophytes in buds of Scots pine.

We studied the location and distribution of a bacterial isolate, a Mycobacterium sp., in buds of Scots pine (Pinus sylvestris L.). Using a probe specific for the 16S rRNA of the Mycobacterium sp., the bacterium was found by in situ hybridization in the meristematic tissues of 40% of all bud samples examined. Because we had previously found other bacterial and fungal endophytes in the meristematic tissues of Scots pine buds, we studied their occurrence in buds during shoot development and dormancy. Using probes targeted to the 16S or 18S rRNA of the endophytes Mycobacterium sp., Methylobacterium spp., Pseudomonas spp. and Rhodotorula minuta, endophytes were found in association with growing tissues, with Methylobacterium spp. being the dominant species. Endophytes were detected in abundance before elongation or differentiation of a bud, but once a tissue was fully developed, endophytes were no longer detected. Metabolic activity of the endophytes was suppressed at the onset of, and during, dormancy of Scots pine, but recovered before the following growing season.

In Situ Hybridization↗

PubMed2004

Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures.

Endophytes are found in meristematic bud tissues of Scots pine (Pinus sylvestris L.) especially prior to growth, which would suggest their involvement in growth of the bud. To test this hypothesis, production of phytohormones by two bacterial (Methylobacterium extorquens, Pseudomonas synxantha) and one fungal endophyte (Rhodotorula minuta) was studied by mass spectrometry. The most common gibberellins, auxins, or cytokinins were not detected in the fractions studied. Instead, M. extorquens and R. minuta produced adenine derivatives that may be used as precursors in cytokinin biosynthesis. A plant tissue culture medium was conditioned with the endophytes, and pine tissue cultures were started on the media. Tetracycline inhibited callus production, which was restored on the endophyte-conditioned media. In addition, conditioning mitigated browning of the Scots pine explants. However, a decrease in tissue size was observed on the endophyte-conditioned media. Addition of adenosine monophosphate in the plant culture medium restored callus production and increased growth of the tissues, but had no effect on browning. Therefore, production of adenine ribosides by endophytes may play some role in the morphological effect observed in the pine tissues.

Journal Article↗

PubMed1999

Identification of Epichloë endophytes in planta by a microsatellite-based PCR fingerprinting assay with automated analysis.

Epichloë endophytes are a group of filamentous fungi that include both sexual (Epichloë) and asexual (Neotyphodium) species. As a group they are genetically diverse and form both antagonistic and mutualistic associations with temperate grasses. We report here on the development of a microsatellite-based PCR system for fingerprinting this group of fungi with template isolated from either culture or infected plant material. M13mp19 partial genomic libraries were constructed for size-fractionated genomic DNA from two endophyte strains. These libraries were screened with a mixture of DIG-labeled dinucleotide and trinucleotide repeat probes. Positive clones were sequenced, and nine unique microsatellite loci were identified. An additional microsatellite was serendipitously identified in the 3' untranscribed region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from N. lolii Lp19. Primers were designed for each locus and a panel of endophytes, from different taxonomic groupings, was screened to determine the degree of polymorphism. On the basis of these results a multiplex assay was developed for strain identification with fluorescently labeled primers for five of these loci. Using this system the size of the products amplified can be precisely determined by automated analysis, and an allele profile for each strain can be readily generated. The assay was shown to resolve endophyte groupings to the level of known isozyme phenotype groupings. In a blind test the assay was used successfully to identify a set of endophytes in planta. A reference database of allele sizes has been established for the panel of endophytes examined, and this will be expanded as new strains are analyzed.

Alleles↗