The contamination of a perennial ryegrass (Lolium perenne) dairy pasture infected with a novel endophyte with other ryegrass/endophyte associations was studied over 2 years in Hamilton, New Zealand. Five management treatments (turnip crop, usual rotational grazing, usual rotational Grazing followed by topping, silage making, hay making) were applied to produce a range of natural reseeding levels, and then three establishment methods (spray/cultivation, double-spray/fallow, hard-grazing) were used before drilling AR1 endophyte-infected perennial ryegrass (no lolitrem B or ergovaline production). Contamination from wild endophyte (Neotyphodium lolii)-infected perennial ryegrass, endophyte-free perennial ryegrass, wild endophyte-infected hybrid (L. boucheanum syn. L. hybridum)/Italian ryegrass (L. multiflorum), N. occultans endophyte-infected hybrid/Italian ryegrass and endophyte-free hybrid/Italian ryegrass tillers was determined. Contamination was assessed in the two summers following the autumn (May 2000) sowing by measuring lolitrem B concentration in bulk ryegrass samples and by sampling individual ryegrass tillers for endophyte identification in late summer of Year 1. Within the hard-grazed establishment treatment, the percentage of ryegrass tillers infected with AR1 in late summer of Year I was highest (73%) following the turnip crop pre-sowing treatment and lowest (4%) after hay making (SED = 5.3%, P < 0.001). The percentages of AR1-infected perennial ryegrass tillers in pastures established following spray/cultivation and double-spray/fallow treatments were 86 and 76%, respectively, compared with only 15% in the hard-grazed treatment (SED = 2.5, P < 0.001) (turnips excluded). The spray/cultivation and double-spray/fallow establishment treatments consistently had lower concentrations of lolitrem B in bulk ryegrass samples than the hard-grazed treatment, for example in early autumn of Year 1 (0.3, 0.5, 1.1 mg kg(-1) DM, P < 0.001) and Year 2 (0.1, 0.2, 1.0 mg kg(-1) DM, P < 0.001). Pasture production from August 2000 to August 2001 was similar following the spray/cultivation, double-spray/fallow and hard-grazed establishment treatments, averaging 14.9 t DM ha(-1). It is recommended that ryegrass pastures containing a novel endophyte be sown after a summer crop, or following a spray/cultivation or double-spray/fallow establishment method, instead of under-sowing into existing hard-grazed pasture. Methodology for assessing pasture contamination with different ryegrass/endophyte associations is also discussed.

The aim of the two experiments reported here was to determine whether cows grazing perennial ryegrass infected with wild endophyte (Neotyphodium lolii) had reduced plasma prolactin concentrations compared with cows consuming a diet containing zero or low levels of ergovaline. The seasonal profile of plasma prolactin concentration was determined in 27 dairy cows grazing perennial ryegrass infected with wild endophyte, and in 28 cows fed a total mixed ration (TMR) diet in the 1999/2000 season. The concentration of prolactin in blood plasma from cows fed TMR was generally higher than the pasture-fed cows from November to April (20.6 versus 16.9 ng ml(-1), standard error of difference (SED) = 1.13), but the magnitude of the difference changed over time. Peak ergovaline levels of 0.8 mg kg(-1) were recorded in ryegrass pasture in mid March 2000. In a second experiment, 18 cows grazing perennial ryegrass infected with the novel endophyte AR1 (no ergovaline or lolitrem B production) had higher concentrations of prolactin in February 2001 than 18 cows grazing ryegrass infected with wild endophyte (25.2 versus 14.4 ng ml(-1), SED = 2.66), although concentrations were similar during January 2001. Prolactin concentration does not provide a reliable indicator of endophyte effects in dairy cows.

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.

Argentine stem weevil damage was assessed in 1998 and 1999 on two diploid perennial ryegrasses (cvs. 'Yatsyn' and 'Samson'), a diploid long-rotation hybrid ryegrass (cv. 'Marsden'), a tetraploid perennial ryegrass selection ('4N') and in a tetraploid hybrid (cv. 'Greenstone') in a field trial in Taranaki. 'Greenstone' was infected with a selected Neotyphodium endophyte that produces peramine and ergovaline but not lolitrem B. All other cultivars were naturally infected with endophytes that produce all three alkaloids. In the first year, percent endophyte infection levels were low in 'Greenstone' (61%), 'Marsden' (70%) and '4N' (71%) relative to 'Yatsyn' (85%) and 'Samson' (86%), but increased in the second year to greater than 88% in all cultivars. Argentine stem weevil adult feeding and larval damage were greatest on 'Greenstone' and least on 'Yatsyn' and 'Samson' in both years. 'Greenstone' tillers damaged by larvae were more severely affected than tillers of other cultivars. Individually and in combination, percent endophyte and peramine and lolitrem B levels in the leaf lamina were correlated with adult activity which in turn was the main factor affecting larval damage. Overall, the tetraploid and hybrid species sustained more damage than the diploids and perennials. This was attributed mainly to lower leaf lamina peramine concentrations in the hybrid and tetraploid combinations which affected adult feeding, but additional attractiveness factors in these cultivars may also play a role in their susceptibility to Argentine stem weevil. The absence of lolitrem B was not a major factor in the number of tillers damaged by larvae in 'Greenstone', but may have resulted in more severe damage to those tillers.

Endophytic fungi in pasture grasses produce alkaloids which affect invertebrate and vertebrate herbivores. While the competence to produce an alkaloid is a property of the fungus, the host plant may moderate fungal activity. Host genetic influence on endophyte activity was studied in perennial ryegrass (Lolium perenne L.) infected with a common strain of Neotyphodium lolii (Latch, Christensen & Samuels) Glenn, Bacon & Hanlin. Progeny seedling families of a partial diallel cross and their 12 parent clones were compared in a glasshouse experiment. Peramine and ergovaline concentrations were determined by high pressure liquid chromatography (HPLC), and intensity of endophyte infection was determined by enzyme-linked immunosorbent assay (ELISA). Concentrations of peramine and ergovaline and the amount of endophyte mycelium in plants varied between families, consistently across two glasshouse cells and (for the HPLC data) two harvests. There was no indication of any maternal effects. Host genetic control was evident in significant general combining ability effects and smaller specific combining ability effects. Parent-progeny correlation coefficients were high, and narrow-sense heritability was estimated as 0.70, 0.72, and 0.58 respectively for ergovaline, peramine, and ELISA. Further analysis indicated little interaction between loci, and no directional dominance. The three traits were correlated, indicating that 41 and 65% of the genetically controlled variation in ergovaline and peramine concentrations, respectively, was a function of mycelial mass. However, there were departures from these relationships. Host plant selection may enable development of pastures with controlled low levels of toxic but ecologically beneficial endophyte metabolites.

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.

ABSTRACT 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.

Two glasshouse experiments were conducted to evaluate possible allelopathic effects of perennial ryegrass/endophyte (Lolium perenne/Neotyphodium lolii) associations on white clover (Trifolium repens). The influence of between-species competition, environmental stress, and soil fauna was eliminated by the use of a sterile sand nutrient culture technique to sustain potted clover seedlings, to which aqueous extracts from moisture-stressed perennial ryegrass pseudostem were applied. In Experiment 1, extracts from two ryegrass cultivars ('Grasslands Pacific' and 'Grasslands Nui'), each hosting in separate seed lines two endophyte strains (ES, WT), suppressed clover growth (mean suppression 22% at 100% concentration) relative to extracts from endophyte-free lines of the same cultivars. The degree of suppression increased with increasing extract concentration, differed between cultivars, but did not differ between endophyte strains. In Experiment 2, extracts at 100% concentration from a third ryegrass cultivar ('Grasslands Ruanui'), hosting in separate seed lines three endophyte strains (AR4, ES, WT), suppressed clover growth by a mean of 27% relative to extracts from an endophyte-free line of this cultivar (range 11 to 47%), with significant differences between strains. The endophyte strains differed in three known endophyte alkaloids, viz peramine, ergovaline, lolitrem B, but in this study none of these alkaloids could solely account for the allelopathic response. The suppression of clover by all ryegrass cultivar/endophyte strain associations tested indicates that clover suppression is not confined to the specific associations used in previous studies, and that further testing of new associations is warranted as variation in the degree of allelopathy existed, being dependant on endophyte strain and host ryegrass cultivar.

Leaf blades of tall fescue (Festuca arundinacea) plants infected with the endophytic fungus Neotyphodium coenophialum (= Acremonium coenophialum) have often been assumed to be endophyte-free. However, fungal colonies identified as N. coenophialum developed following incubation of surface-sterilised leaf blades from endophyte-infected tall fescues on antibiotic potato dextrose agar. A standardised fungal isolation technique was developed to assess the distribution of hyphae within tall fescue leaf blades in three USA-bred cultivars ('Georgia 5', 'Kentucky 31', 'Jesup') and a roadside population (MRSP) from the Manawatu region, New Zealand. Leaf blades of the three USA-bred cultivars were typically sparsely colonised. In contrast, extensively colonised leaf blades were present in many MRSP plants, particularly in autumn. Ergovaline concentrations of up to 3.9 ppm (mean 2.5) were present in leaf blades of the MRSP grown in a glasshouse. Ergovaline at lower concentrations (mean 0.4 ppm) was also detected in leaf blades of the tall fescue cultivars infected with their natural endophytes. These unusually high concentrations of hyphae and ergovaline observed within leaf blades of the MRSP provide an explanation of why fescue toxicosis has been especially severe in New Zealand. Significantly, N. coenophialum from plants of the MRSP was not uniform in cultural characteristics and conidial length, or in the ability to colonise leaf blades and produce ergovaline in artificially infected plants of four diverse tall fescue cultivars. The four inoculated cultivars ('Georgia 5', 'Grasslands Roa', 'Kentucky 31', 'KFa949') also differed in their interaction with N, coenophialum from MRSP. The most colonised leaf blades were in plants from an experimental cultivar of Mediterranean origin (KFa949), and, surprisingly, ergovaline concentrations were higher in the leaf blades than sheath. The New Zealand-bred tall fescue cultivar 'Grasslands Roa' was also distinctive in that no hyphae were detected in leaf blades. The concentration of ergovaline present in pooled leaf blades of 'Grasslands Roa' plants was lower than in those of the other naturally and artificially infected tall fescue/N. coenophialum populationsexamined in this study, reflecting the apparently low concentrations of hyphae present.

In the course of measurements of alkaloid concentrations in several thousand samples of endophyte-infected perennial ryegrass, tall fescue, and meadow fescue plants for diverse studies, we have encountered a number of samples with alkaloid concentrations which greatly exceed the highest levels reported in previous studies (e.g. Welty et al., 1994).

A preliminary study of the effect of water stress on alkaloid concentrations in endophyte-infected perennial ryegrass (Lolium perenne) focused on artificial infections with selected endophyte strains (Barker et al., 1993). This paper reports the results of studies of the impact of environmental factors (nitrogen, water, temperature) on the concentration of alkaloids in "Nui" perennial ryegrass naturally infected with Neotyphodium lolii, a more typical component of New Zealand pastoral forage.

Hyphae of Neotyphodium coenophialum are generally considered to be absent or insignificant within leaf blades of tall fescue. However, a recent study found hyphae in leaf blade tissue close to the ligule in nearly all tall fescue plants examined, while in some plants hyphae were abundant along the entire length of blade. (paper prepared). This short paper reports on a study which examined the influence of plant and endophyte genotype on leaf blade colonization, and ergovaline distribution within leaves.

Ryegrass tillers, from a ryegrass staggers-prone pasture, were dissected into constituent live leaves of different ages. Leaves were further divided into three blade and two sheath portions. Acremonium lolii, lolitrem B, and peramine concentrations were determined for each defined portion. The mean % distribution and concentrations of A. lolii and lolitrem B in the sheath were much higher, and the mean % distribution of peramine in the sheath was much lower, than in the blade portions. Within leaves, both the % distribution and the concentration of A. lolii increased progressively from blade tip to lower sheath; the patterns for % distribution and concentration of lolitrem B were similar to those for A. lolii, except that lower sheath values were less than those of upper sheath portions; the % distribution of peramine was higher in the lower blade portions than elsewhere, and peramine concentrations were higher in portions adjacent to the ligule. Within tillers, A. lolii concentrations were lower in the youngest and oldest leaves; peramine concentrations were lower for older than for younger leaves; and lolitrem B concentrations increased progressively from youngest to oldest leaves.

Isozyme analysis, morphological characters, sensitivity to benomyl in vitro, and the production of alkaloids in the fungal-host-grass associations were used to examine the relationships between Acremonium endophytes of Festuca arundinacea, F. pratensis and Lolium perenne. Isozyme analysis identified six taxonomic groupings, with each grouping comprising isolates from just one of the three grass species. Three taxonomic groupings occurred in F. arundinacea, two in L. perenne, and one in F. pratensis. Alkaloid production in the natural host grass-Acremonium associations proved a useful taxonomic criterion, with the profile of alkaloids being consistent in the host plants for all isolates within a single isozyme phenotype and for most isolates within a taxonomic grouping. On the basis of morphological, cultural and secondary product criteria, only one of the three taxonomic groupings identified amongst the F. arundinacea endophytes fitted the definition of A. coenophialum, and only one grouping of L. perenne endophytes could be accommodated within A. lolii. All isolates from F. pratensis could be accommodated within A. uncinatum even though the conidia differed between isolates of the two isozyme phenotypes identified during this study.

Endophytes from perennial rye-grass (Lolium perenne) infected with fungi having intercellular mycelium characteristic of Acremonium lolii exhibited wide variation in cultural, morphological and physiological characteristics. Most isolates could be accommodated within the taxon A. lolii, although they were unstable in culture and had greater variation than previously described. Some, however, resembled A. typhinum, the anamorph of Epichloe typhina, but symptoms of choke disease were not observed. The variation observed within isolates indicates the need for reappraisal of the taxonomy of Acremonium sect. Albo-lanosa.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAn Efficient Method for the Isolation of Peramine, an Insect Feeding Deterrent Produced by the Fungus Acremonium LoliiDaryl D. Rowan and Brian A. TapperCite this: J. Nat. Prod. 1989, 52, 1, 193–195Publication Date (Print):January 1, 1989Publication History Published online1 July 2004Published inissue 1 January 1989https://pubs.acs.org/doi/10.1021/np50061a030https://doi.org/10.1021/np50061a030research-articleACS PublicationsRequest reuse permissionsArticle Views179Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Two methods are described for the detection and measurement of peramine, an alkaloid from endophyte-infected grasses with insect feeding deterrent activity. Both procedures involve ion-exchange work up of extracts followed by either thin-layer chromatography with detection by Ehrlich's reagent or quantitation by reversed-phase high-performance liquid chromatography with UV detection at 280 nm.

The photosensitisation of young albino rats fed diets containing specially prepared lucerne leaf protein concentrates (LPC) or purified pigments has been investigated. Signs of intense photosensitivity including death occurred after a few hours of illumination in animals fed a diet containing 20% LPC prepared by holding lucerne juice at 70 °C for 55 min. However a sample prepared by briefly heating juice to 90 °C was not active. Mixed chlorophyllides and purified pheophorbide a were strongly photosensitising whereas pheophorbide b was much less active and pheophytin a was inactive. Where strong photosensitisation occurred pigments derived from chlorophyll could be extracted from blood plasma and livers. Examination of stomach and intestine contents showed that dietary chlorophyllides and chlorophylls were converted to pheophorbides and pheophytins in the gut. The photosensitising potential of a diet can be related principally to the combined content of pheophorbide a and chlorophyllide a.

Glyoxylic acid cyanohydrin was found to be the principal cyanogen accumulating in cultures of snow mold, type B, strain W2, grown on media containing yeast and malt extracts. Small amounts of pyruvic acid cyanohydrin were also present. There was no evidence for presence of linamarin or lotaustralin in the cultures.