Epoxyjanthitrems I-IV (1-4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungusEpichloe festucaevar.lolii.Although structures for epoxyjanthitrems I-IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests.

Epoxyjanthitrems I–IV (1–4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichloë festucae var. lolii. Although structures for epoxyjanthitrems I–IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests.

Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. Many Epichloë species have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterize Epichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. The Epichloë species so far identified as the closest known relatives of the two progenitors of E. hybrida are E. festucae var. lolii and E. typhina. We confirm that the nuclear genome of E. hybrida contains two homeologs of most protein-coding genes from E. festucae and E. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from the E. typhina subgenome. Mitochondrial DNA is detectable only from E. festucae, whereas ribosomal DNA is detectable only from E. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally in E. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers.

Polyclonal antibodies were produced in sheep for the development of a competitive enzyme-linked immunoassay for use in quantifying loline alkaloids in pasture samples. Lolines are aminopyrrolizidine secondary metabolites produced by fungal endophytes present in tall fescue and meadow fescue grasses, and confer increased resistance to a range of grass pests. Immunizing and plate coating antigens were prepared with derivatized loline dihydrochloride. Cross-reactivity studies indicated that the assay developed has broad specificity and antibody binding to loline analogues is affected by structural changes in the side chain of the loline molecule. Results obtained using the optimized immunoassay were compared with those determined by gas chromatography. The assay provides a sensitive and rapid analytical method that detects the loline analogues of interest and has been applied in pasture breeding programmes. The assay limit of quantitation for lolines in pasture is 3 mu g/g in dried herbage.

The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi.

Turnip (Brassica rapa ssp. rapa) and rape (Brassica napus ssp. biennis) and other brassica forage crops are regarded as "safe" feed for cattle during late summer and fall in the North Island of New Zealand when high Pithomyces chartarum spore counts in pastures frequently lead to sporidesmin toxicity (facial eczema). Sporadic acute severe cases of turnip photosensitization in dairy cows characteristically exhibit high γ-glutamyl transferase and glutamate dehydrogenase serum enzyme activities that mimic those seen in facial eczema. The two diseases can, however, be distinguished by histopathology of the liver, where lesions, in particular those affecting small bile ducts, differ. To date, the hepato-/cholangiotoxic phytochemical causing liver damage in turnip photosensitization in cattle is unknown. Of the hydrolysis products of the various glucosinolate secondary compounds found in high concentrations in turnip and rape, work has shown that nitriles and epithionitriles can be hepatotoxic (and nephro- or pancreatotoxic) in rats. These derivatives include β-hydroxy-thiiranepropanenitrile and 3-hydroxy-4-pentenenitrile from progoitrin; thiiranepropanenitrile and 4-pentenenitrile from gluconapin; thiiranebutanenitrile and 5-hexenenitrile from glucobrassicanapin; phenyl-3-propanenitrile from gluconasturtiin; and indole-3-acetonitrile from glucobrassicin. This perspective explores the possibility of the preferential formation of such derivatives, especially the epithionitriles, in acidic conditions in the bovine rumen, followed by absorption, hepatotoxicity, and secondary photosensitization.

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea, respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fungal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed.

Epichloae endophytes of family Clavicipitaceae (comprising genera Epichloë and Neotyphodium ) are fungal symbionts of Pooideae grasses. The associations formed, range from mutually beneficial to antagonistic and the nature of this relationship is dependent upon the importance of vertical (via host seeds) versus horizontal (ascospore mediated) transmission of the fungus. These endophytes can enhance their hosts’ survival through protection from abiotic and biotic stresses and can thus be utilized in an agricultural context. Animal-safe grass-endophyte associations that confer bio-protective properties for increased pasture persistence and productivity have been developed and commercialized. One of the crucial drivers underpinning the selection of epichloae strains for commercial development is endophyte derived bioactivity. The potential of next generation endophytes is determined by testing a number of attributes such as agronomic fitness, animal and food safety as well as compatibility with host plants of interest. Strategic research supports these activities by focusing on elucidating mechanisms of compatibility between host and fungal symbiont, as well as investigating other molecular drivers of symbiosis such as siderophore mediated iron-uptake, fungal signalling, fungal growth in host plants and fungal secondary metabolism. This review weaves together the different strands of multidisciplinary research aimed at ultimately exploiting epichloae endophytes for increased pasture performance.

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole‐diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub‐telomeric locus in the genome of E. festucae . Here we resolve the genetic basis for the remarkable indole‐diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.

ABSTRACTBioprotective alkaloids produced byEpichloëand closely related asexualNeotyphodiumfungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. TheLTMlocus ofNeotyphodium lolii(Lp19) andEpichloë festuce(Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10ltmgenes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10ltmgenes were present in only three epichloë endophytes. A predominance of the asexualNeotyphodiumspp. examined contained 8 of the 10ltmgenes, with only oneN. loliicontaining the entireLTMlocus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present inltmclusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.

Bioprotective alkaloids produced by Epichloë and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloë festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloë endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.

While it is generally accepted that Neotyphodium lolii and related epichloë endophytes are mutualists that provide important benefits to their perennial ryegrass (Lolium perenne) host plants under conditions of biotic stress, relatively little is known about the effect of endophyte on the host under conditions of abiotic stress. Using genetically identical endophyte infected (E+) and uninfected (E−) clones of perennial ryegrass from a natural and a synthetic association grown under conditions of water stress in a controlled environment, we show that N. lolii had minor effects on morphological responses (leaf elongation rate and ground biomass production) but had more pronounced effects on physiological responses (water use efficiency, relative water content and osmotic potential) by the host. The effects were most marked in the natural association. While levels of proline increased in response to water stress, the presence of endophyte had no effect on those levels. The effect of water stress on endophyte bioprotective metabolites was also examined. Ergovaline levels in pseudostem tissue increased in response to increasing water stress for both N. lolii/L. perenne associations but lolitrem B levels only increased in the natural association. No differences in steady state levels of transcripts from genes known to be required for the synthesis of these alkaloids were observed in response to water stress. This study demonstrated that N. lolii can confer protection to perennial ryegrass from water stress and that levels of the bioprotective metabolites, lolitrem B and ergovaline were altered in response to this abiotic stress in a manner that was specific for each symbiotic association.

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.

A fast method was developed to directly infuse raw plant extracts into a linear ion trap mass spectrometer, using the ion trap to isolate and fragment as many ions as possible from the extract. The full mass spectra can be analysed by multivariate statistics to determine discriminating ions, and the fragmentation data allows rapid classification or identification of these ions. The methodology was used to screen a wide range of strains of endophytic fungi in perennial ryegrass seeds for differences in metabolic profiles. The results show that this newly developed methodology is able to determine discriminating ions that can be present in very low concentrations. It also yielded sufficient fragmentation data to classify or identify the discriminating ions.

Three grass host species—tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.—each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.

Peramine concentrations in a range of ryegrass/ AR1 endophyte associations have been measured during pre-market safety testing. While concentrations for diploid perennial ryegrass cultivars were in excess of 15ppm a small number of associations were below 10ppm. These proved to be tetraploid ryegrass. When assessing the effectiveness of a ryegrass/AR1 association to deter ASW, and other invertebrate pasture pests, the host ryegrass cultivar or genotype and consequent peramine concentrations need to be taken into consideration.

This chapter contains section titled: Arl is Now the Benchmark in New Zealand Tall Fescue Endophyten in New Zealand Further Research with Ryegrass Seed Inventory and Quality Control Endophyte Genetic Research Native Grasses Control of Vertebrate Pests Current Priorities References