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A PCR and Southern analysis method was used to screen a broad taxonomic range of 44 endophyte isolates to determine the differences related to indole-diterpenes which are present in few endophyte-grass association compared to that of the other bioprotective alkaloids, which are more widespread among the endophytes. Analysis of toxin biosynthesis genes by PCR allows a diagnostic method to screen endopytes for both beneficial and detrimental alkaloids and could be applied as a resource for screening isolates required for forage improvement.

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 µg/g in dried herbage. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A microsatellite-based polymerase chain reaction (PCR) DNA fingerprint assay for the identification of Epichloe endotypes in culture and in planta has been developed. Epichloe endotypes are a group of filamentous fungi with sexual and asexual species. Identifying the endotype strains was carried out by constructing M13mp19 partial genomic libraries for size-fractionated genomic DNA. The libraries were then screened with a mixture of DIG-labeled dinucleotide and trinucleotide probes. Nine unique microsatellite loci were identified after positive clones have been sequenced.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 MS n 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. [Copyright &y& Elsevier]

Neotyphodium sp. Lp1, an endophytic fungus from perennial ryegrass (Lolium perenne), produces the mycotoxin ergovaline in infected grasses, whereas a mutant in which a particular peptide synthetase gene is knocked out does not. We examined the impact of this knockout on other constituents of the ergot alkaloid pathway. Two simple lysergic acid amides, ergine and a previously undescribed amide, were eliminated by the knockout. Lysergic acid accumulated in the knockout endophyte, but quantities were only 13% of the total lysergic acid derivatives accumulated in the wild type. Concentrations of several clavines were not substantially affected. However, a novel clavine accumulated to higher concentrations in perennial ryegrass containing the knockout strain. The results indicate that production of simple lysergic acid amides requires the activity or products of the ergovaline-associated peptide synthetase and that the regulation of ergot alkaloid production is modified in response to the relatively late block in the pathway.

An improved extraction and cleanup procedure for quantitative analysis of ergovaline in Neotyphodium-infected grass tissues by high-performance liquid chromatography was developed, utilizing aqueous 2-propanol-lactic acid as extraction solvent. Losses of sample material and time requirements were significantly reduced, handling procedures simplified, and ergovaline and internal standard ergotamine recovered with similar efficiency from extracts. Analyses can be carried out on very small amounts (2-5 mg of dry weight) of samples and another endophyte-alkaloid, peramine, determined in the same extracts. Calibration curves with 2-propanol-lactic acid were linear over the range 0.004-0.938 microM ergovaline (= 2-500 ng/mL) in extracts, corresponding to 0.04-10 microg/g in samples. The distribution of ergovaline in the plant was extremely heterogeneous, indicating low in-planta mobility and strong regulation of accumulation by the internal plant environment. In contrast, peramine was much more uniformly distributed. These results clearly demonstrate very large differences in the tissue specificities of ergovaline and peramine.