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Simple Summary: This study investigates the impact of endophyte-infected ryegrass and the alkaloids (such as peramine) which it produces, on the Argentine stem weevil (Listronotus bonariensis), a pest affecting pastures crucial for extensive agriculture. Seven unnamed lines (LE161-LE167), and two Lolium perenne cultivars, Jumbo and Alto AR1, were evaluated. Leaves from LE164, LE166, and ALTO AR1 led to significant weight reduction in L. bonariensis (−13.3%, −17.1%, and −18.2%, respectively). The corresponding alkaloidal extracts from these lines, grown in greenhouse trials, exhibited an antifeedant effect in laboratory assays, resulting in reduced weevil weight (−12.5%, 8.8%, and 4.9%, respectively). When incorporated into an artificial diet, peramine induced an antifeedant effect. Liquid chromatography analysis revealed the presence of peramine in LE164, LE166, and ALTO AR1 (ranging from 46.5 to 184.2 ng g−1), while it was absent in Jumbo and other lines. These findings suggest that endophyte-induced peramine production in specific ryegrass lines negatively impacts the feeding performance of L. bonariensis, presenting a potential ecological approach for pest control in pastures crucial for extensive agriculture. One of the primary supports for extensive agriculture is pasture, which can suffer severe damage from insects including the Argentine stem weevil, Listronotus bonariensis. The main control method has been the infection of ryegrass with an endophyte fungus, forming a symbiotic association that produces alkaloids. In this study, we evaluated the impact of endophyte and peramine production on the weight of L. bonariensis across seven unnamed lines (LE161-LE167), and two Lolium perenne cultivars: Jumbo and Alto AR1. L. bonariensis adults fed on leaves from LE164, LE166, and ALTO AR1 showed weight losses of 13.3%, 17.1% and 18.2%, respectively. Similarly, the corresponding alkaloidal extract from LE164, LE166, and ALTO AR1 exhibited an antifeedant effect on L. bonariensis adults in laboratory assays, as observed through weight loss or low weight gain (−12.5%, 8.8% and 4.9%, respectively). Furthermore, one alkaloid, peramine, also elicited an antifeedant effect when incorporated into an artificial diet. Liquid chromatographic analysis of the alkaloid extract revealed that peramine was present in LE164, LE166 and ALTO AR1 in amounts ranging from 46.5–184.2 ng/g. Peramine was not detected in Jumbo and the remaining experimental lines. These data suggest that L. bonariensis were susceptible to peramine produced from endophyte infection in experimental lines LE164 and LE166, as well as ALTO AR1, affecting their feeding behavior. [ABSTRACT FROM AUTHOR]

Both adults and larvae of Listronotus bonariensis damage ryegrass (Lolium spp.), a dominant component of New Zealand pasture. Damage can be reduced by Epichloë festucae var. lolii, a biotrophic fungal symbiont of ryegrass, through the production of alkaloids with deterrent and/or toxic effects to insect herbivores including L. bonariensis. Here we report results from two pot trials and a field trial comparing endophyte strains with different alkaloid profiles that have been introduced to reduce or eliminate adverse effects on stock. Adult L. bonariensis feeding scars, number of eggs and the proportion of tillers with larval damage and the severity of that damage were recorded. Samples of pseudostem and leaf blades were analysed for alkaloids. AR1 endophyte reduced adult feeding, oviposition, and larval damage whereas AR37 had no effect on adults but was highly effective in reducing larval damage. Reduction in adult feeding and oviposition by different NEA strains was governed by concentrations of peramine and ergovaline. For plants infected with AR37, alkaloid concentrations that determined the extent of larval damage were influenced by host species and ploidy; tetraploid L. perenne was more severely damaged than its diploid equivalent; L. multiflorum cultivars were more susceptible than the L. hybridum and L. perenne cultivars. [ABSTRACT FROM AUTHOR]

One of the primary supports for extensive agriculture is pasture, which can suffer severe damage from insects including the Argentine stem weevil, Listronotus bonariensis. The main control method has been the infection of ryegrass with an endophyte fungus, forming a symbiotic association that produces alkaloids. In this study, we evaluated the impact of endophyte and peramine production on the weight of L. bonariensis across seven unnamed lines (LE161-LE167), and two Lolium perenne cultivars: Jumbo and Alto AR1. L. bonariensis adults fed on leaves from LE164, LE166, and ALTO AR1 showed weight losses of 13.3%, 17.1% and 18.2%, respectively. Similarly, the corresponding alkaloidal extract from LE164, LE166, and ALTO AR1 exhibited an antifeedant effect on L. bonariensis adults in laboratory assays, as observed through weight loss or low weight gain (−12.5%, 8.8% and 4.9%, respectively). Furthermore, one alkaloid, peramine, also elicited an antifeedant effect when incorporated into an artificial diet. Liquid chromatographic analysis of the alkaloid extract revealed that peramine was present in LE164, LE166 and ALTO AR1 in amounts ranging from 46.5–184.2 ng/g. Peramine was not detected in Jumbo and the remaining experimental lines. These data suggest that L. bonariensis were susceptible to peramine produced from endophyte infection in experimental lines LE164 and LE166, as well as ALTO AR1, affecting their feeding behavior.

Cool season grasses often form reciprocal symbiotic relationships with endophytic fungal species in genus Epichloë. In this study, we characterized three fungal endophytes isolated from the grass Hordeum bogdanii native to northwest China. Based on morphological characteristics and phylogenetic analyses of tefA, tubB, and actG sequences, we identified them as Epichloë sp. HboTG-2 (H. bogdanii Taxonomic Group 2: E. bromicola × E. typhina). Alkaloid synthesis related genes analysis showed that Epichloë sp. HboTG-2 may have the ability only to produce peramine which is toxic to insects but not to animals. In the process of this study, we did not observe sexual structures or epiphyllous growth on leaves of infected plants. [ABSTRACT FROM AUTHOR]

Epichloë endophytes in grass associations express a myriad of secondary metabolites which can affect the health of grazing animals and reduce the impact of insect pests on pasture. The ideal endophyte-grass association must have a favourable chemical profile such that the impact on animal health is minimised while the beneficial, deterrent effect on insect pests is maximised. A number of endophyte-perennial ryegrass associations have been successfully commercialised but research is on-going to further improve production in farming systems. Secondary metabolites expressed by endophyte-infected tall fescue include lolines, an animal-safe class of compound which imparts a potent effect on insects. Since endophyte-infected perennial ryegrass does not express lolines, a tall fescue endophyte, AR501, was inoculated into perennial ryegrass in an attempt to improve the insect resistance of this pasture type. In addition to animal safety, it is imperative that consideration is given to the safety of humans consuming animal products derived from livestock grazing the novel pasture. Although pure loline alkaloids have previously been tested on mice it is essential that the entire AR501 endophyte-infected perennial ryegrass matrix is tested since this will result in the exposure of both known and unknown secondary metabolites to mice. Three treatment groups each containing 6 male and 6 female mice were fed diets containing AR501 endophyte-infected perennial ryegrass seed (30%), perennial ryegrass seed containing no endophyte (30%) or a diet without seed (control) for 3 weeks. Mice fed control diet ate more than either of the treatment groups fed a diet containing seed. Male mice fed diet containing Nil endophyte seed ate more than those eating AR501 endophyte-infected perennial ryegrass seed although there was no difference observed in the food intake of female mice. While a few statistically significant differences were observed in the haematology and serum biochemical data, in every instance the difference was restricted to only one gender so is considered unlikely to be of toxicological significance. Mice fed AR501 endophyte-infected perennial ryegrass seed remained healthy throughout the experimental period despite consuming 62,000 mg/kg lolines and 4600 mg/kg peramine per day as well as the wide array of other unknown secondary metabolites expressed by this endophyte. Although animal products may contain additional metabolites as a result of animal metabolism, this experiment raises no food safety concerns for AR501 endophyte-infected perennial ryegrass. [Display omitted] • Endophytes are used to confer beneficial insect pest protection to their host plants. • Endophytes produce a myriad of known and unknown secondary metabolites. • Secondary metabolites consumed by animals could go up the food chain to be consumed by humans. • AR501 endophyte-infected ryegrass seed was fed to mice for 21 days. • The lack of toxicity observed raises no food safety concerns for these compounds. [ABSTRACT FROM AUTHOR]

Context: Infecting grasses with an Epichloë endophyte contribute significantly to host plant salt tolerance. The Epichloë endophyte alkaloid peramine is primarily responsible for feeding deterrence against some herbivorous insects. Infection by an Epichloë is required for peramine production in the host grass. Limited information is available regarding the effects of salinity stress on Epichloë endophyte colonisation and associated production of peramine metabolites. Aims and methods: In this study, we determined the effects of NaCl stress on the concentration of endophytic hyphae and peramine in wild barley (Hordeum brevisubulatum (Trin.) Link) seedlings infected with Epichloë bromicola. Key results: Seedings dry weight at 4 weeks declined from 7.05 to 4.27 g/plant over the range, 0–300 mM NaCl. The hyphal concentration, as well as the peramine concentration, increased in response to high NaCl concentrations, hyphae and peramine concentration at 4 weeks increased from 13.29 to 264.60 copies/ng gDNA and from 26.75 to 43.19 μg/g DW, respectively. Peramine concentration was positively correlated with the concentration of hyphae (R 2 = 0.4012, P < 0.001). Conclusions and implications: It seems that peramine concentration was mostly explained by the in - planta variation in endophyte hyphae concentration in wild barley infected with Epichloë species under highly saline conditions. The symbiotic association between an Epichloë endophyte and a grass can result in the production of peramine, an alkaloid that can provide symbiont bioprotective effects against insects. The study showed that concentrations of endophytic hyphae and peramine increased in response to high amount of applied NaCl. Hyphal concentration was a good predictor of peramine concentration. The results have enhanced the knowledge of the application of endophytes to provide pest-resistant pastures under abiotic stress. [ABSTRACT FROM AUTHOR]

Perennial ryegrass (Lolium perenne L.) grows in association with a fungal endophyte Epichloe festucae var. lolii (Latch, Christensen & Samuels) Bacon & Schardl, which produces alkaloids that protect the grass against grazing by mammals and insects. These alkaloids are found in guttation fluid (xylem sap exuded from leaves through special structures known as hydathodes) and have the potential to impact on beneficial invertebrates in pastoral ecosystems. Newly emerged adults of the parasitoid Microctonus aethiopoides Loan (Hymenoptera: Braconidae) were supplied with guttation fluid from pot-grown ryegrasses infected with three different strains of endophyte (standard, AR37, AR1) or no endophyte collected at different times of the year, or water, sucrose solution or no liquid. Longevity was compared when individuals were held in separate vials in controlled environment room at 20oC with 16:8 h light:dark photoperiod. An enzymatic method was used to measure sugars in guttation fluid samples collected on three dates. Guttation fluid from endophyteinfected grasses was found to have no detrimental effect on M. aethiopoides longevity and to contain glucose and fructose. Guttation fluid from AR37-infected ryegrass collected in autumn increased insect longevity compared to water and fluid from standard-type infected ryegrass by 26% and 24% respectively. The lack of available food sources in New Zealand ryegrass-dominant pastures means that guttation fluid from AR37-infected ryegrass in autumn may contribute to M. aethiopoides efficacy as a biocontrol agent through enhanced longevity. [ABSTRACT FROM AUTHOR]

Cool season grasses often form reciprocal symbiotic relationships with endophytic fungal species in genus Epichloë. In this study, we characterized three fungal endophytes isolated from the grass Hordeum bogdanii native to northwest China. Based on morphological characteristics and phylogenetic analyses of tefA, tubB, and actG sequences, we identified them as Epichloë sp. HboTG-2 (H. bogdanii Taxonomic Group 2: E. bromicola × E. typhina). Alkaloid synthesis related genes analysis showed that Epichloë sp. HboTG-2 may have the ability only to produce peramine which is toxic to insects but not to animals. In the process of this study, we did not observe sexual structures or epiphyllous growth on leaves of infected plants.

Many cool-season grasses form permanent, mutualistic symbioses with asexual Epichloë endophytes. These fungal symbionts often perform a protective role within the association as many strains produce secondary metabolites that deter certain mammalian and invertebrate herbivores. Although initially a serious issue for agriculture, due to mammalian toxins that manifested in major animal health issues, selected strains that provide abiotic stress protection to plants with minimal ill effects to livestock are now commercialized and routinely used to enhance pasture performance in many farming systems. These fungal endophytes and their grass hosts have coevolved over millions of years, and it is now generally accepted that most taxonomic groupings of Epichloë are confined to forming compatible associations (i.e., symptomless associations) with related grass genera within a tribe. The most desired compounds associated with Epichloë festucae var. lolii, an endophyte species associated with perennial ryegrass, are peramine and epoxy-janthitrems. No other major secondary metabolites with invertebrate bioactivity have been identified within this association. However, other agriculturally beneficial compounds, such as lolines, have been discovered in related endophyte species that form associations with fescue grasses. A rationale therefore existed to develop novel grass-endophyte associations between loline-producing endophytes originally isolated from tall fescue with elite cultivars of perennial ryegrass to achieve a wider spectrum of insect bioactivity. A suitable loline-producing endophyte strain of Epichloë sp. FaTG-3 was selected and inoculated into perennial ryegrass. We hypothesed that endophyte transmission frequency, endophyte mycelial biomass and endophyte-derived alkaloid production would differ between the original tall fescue host and the artificial association. Consistent with our hypothesis, our data strongly suggest that plant species significantly affected the plant-endophyte association. This effect became more apparent for transmission frequency and endophyte biomass as the plants matured. Overall, the viable endophyte infection frequency was greater in the tall fescue host than in perennial ryegrass, at all sampling dates. Additionally, temperature was found to be a significant factor affecting endophyte transmission frequency, endophyte mycelial biomass and alkaloid production. Implications for the development of novel grass-endophyte associations are discussed.

Hordeum vulgare is an important source of feed and forage for livestock, and of food and drink for humans, but its utilization rate is lower than that of other cereal crops, thus it is crucial to improve barley agronomic traits and production. Epichloë bromicola is an endophyte that was isolated from wild barley (Hordeum brevisubulatum). Previous studies have found that Epichloë can indirectly influence the growth of host plants by affecting soil chemical characteristics, the microbial community, and by producing a range of secondary metabolites. However, underlying effects of Epichloë on the abundance and diversity of soil and root microbes have not been well-studied. In addition, there is a question regarding the relationship between endophyte-produced alkaloids and effects on the root and rhizosphere microbial communities. The objective of this study was to investigate changes in agronomic traits, nutritional properties, peramine, soil chemical and microbial community in the fourth generation of new barley symbionts EI (E. bromicola-infection) and EF (E. bromicola-free) in LQ+4 and LZ+4. We understand the plant height and biomass of EI in LZ+4 were significantly higher than those of EF. The HPLC analysis showed that the peramine content of EI in LQ+4 and LZ+4 was 0.085 and 0.1 mg/g, respectively. We compared the bacterial and fungal communities by analyzing the 16s rRNA (for bacteria) and ITS rDNA regions (for fungi). Our data revealed that the composition of fungal communities in rhizosphere soil of LZ+4 EI are higher than EF. In addition, the diversity and richness of fungal communities in root and rhizosphere soil of LQ+4 EI and LZ+4 EI are significantly higher than EF. Rhizosphere soil microbial community composition was higher than that in roots in LQ+4 and LZ+4. Peramine was significantly and positively correlated with the richness of the soil fungal community. Moreover, the principal component analysis (PCoA) results indicated that E. bromicola significantly influenced the community composition of root and rhizosphere soil microbes in both LQ+4 and LZ+4. Our results illustrate that E. bromicola can influence barley growth, peramine production and microbial communities associated with barley.

Many cool-season grasses form permanent, mutualistic symbioses with asexual Epichloë endophytes. These fungal symbionts often perform a protective role within the association as many strains produce secondary metabolites that deter certain mammalian and invertebrate herbivores. Although initially a serious issue for agriculture, due to mammalian toxins that manifested in major animal health issues, selected strains that provide abiotic stress protection to plants with minimal ill effects to livestock are now commercialized and routinely used to enhance pasture performance in many farming systems. These fungal endophytes and their grass hosts have coevolved over millions of years, and it is now generally accepted that most taxonomic groupings of Epichloë are confined to forming compatible associations (i.e., symptomless associations) with related grass genera within a tribe. The most desired compounds associated with Epichloë festucae var. lolii , an endophyte species associated with perennial ryegrass, are peramine and epoxy-janthitrems. No other major secondary metabolites with invertebrate bioactivity have been identified within this association. However, other agriculturally beneficial compounds, such as lolines, have been discovered in related endophyte species that form associations with fescue grasses. A rationale therefore existed to develop novel grass-endophyte associations between loline-producing endophytes originally isolated from tall fescue with elite cultivars of perennial ryegrass to achieve a wider spectrum of insect bioactivity. A suitable loline-producing endophyte strain of Epichloë sp. FaTG-3 was selected and inoculated into perennial ryegrass. We hypothesed that endophyte transmission frequency, endophyte mycelial biomass and endophyte-derived alkaloid production would differ between the original tall fescue host and the artificial association. Consistent with our hypothesis, our data strongly suggest that plant species significantly affected the plant-endophyte association. This effect became more apparent for transmission frequency and endophyte biomass as the plants matured. Overall, the viable endophyte infection frequency was greater in the tall fescue host than in perennial ryegrass, at all sampling dates. Additionally, temperature was found to be a significant factor affecting endophyte transmission frequency, endophyte mycelial biomass and alkaloid production. Implications for the development of novel grass-endophyte associations are discussed. [ABSTRACT FROM AUTHOR]

The distribution of different Epichloë species within eight natural populations of Lolium perenne was studied. In total, 40.2% of the asymptomatic plants were infected by Epichloë festucae var. lolii or by Epichloë typhina. Both species occurred in sympatry in seven grass populations, and some plants had dual infections by both taxa. No hybrid taxa such as Epichloë hybrida were detected. Epichloë festucae strains were classified into two morphotypes, M1 and M3, according to culture characters, both morphotypes occurred in sympatry in seven populations. Plants bearing stromata produced by Epichloë typhina were observed, but asymptomatic plants infected by this species also occurred in seven populations. The alkaloid profile of Lolium perenne plants was related to the morphotype of their infecting strains: most plants infected by M3-strains were characterized by lolitrem, and those with M1-strains contained either ergovaline or lolitrem. Plants infected by Epichloë typhina were characterized by high peramine content. [ABSTRACT FROM AUTHOR]

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains. [ABSTRACT FROM AUTHOR]

The total synthesis of peramine, a natural product isolated from an endophytic fungi, has been achieved in four steps and 34% overall yield from known compounds. The key step was the one-pot construction of the pyrrolopyrazinone ring from pyrrole amide and propargyl bromide. The preparation of peramine-d4 as an internal standard for quantitative analysis by MS is also described. [ABSTRACT FROM AUTHOR]

Alkaloids produced by endophytic fungi can have an important influence on agricultural ecology, and can often be affected by climatic factors. At present, there are no studies that have assessed the relationship between alkaloid production and elevation or climatic factors in the Qinghai-Tibetan Plateau. To address this knowledge gap, we explored ergot alkaloid and peramine production in Epichloë-infected (E+) Elymus tangutorum collected from the Qinghai-Tibetan plateau and assessed the relationship between the concentration of these alkaloids and elevation. The effects of temperature and precipitation on these relationships were also investigated. The concentrations of ergonovine, ergine, and peramine ranged from 0.47–0.84, 0.35–1.72 and 9.18–13.00 µg·g−1, respectively. Fitted cubic regression curves describing an arc-pattern across the elevational series were statistically significant for ergine and peramine concentrations. The elevational trend in peramine concentration was linked to mean daily temperature, while the ergine elevational trend was linked to mean precipitation. Our results provide a new understanding of the climatic factors that influence the alkaloid production of endophytic fungi at different elevations in the Qinghai-Tibetan plateau.

Codon usage bias plays an important role in shaping genomes and genes in unicellular species and multicellular species. Here, we first analyzed codon usage bias in seven Epichloë species and their peramine-coding genes. Our results showed that both natural selection and mutation pressure played a role in forming codon usage bias in seven Epichloë species. All seven Epichloë species contained a peramine-coding gene cluster. Interestingly, codon usage bias of peramine-coding genes were not affected by natural selection or mutation pressure. There were 13 codons more frequently found in Epichloë genome sequences, peramine-coding gene clusters and orthologous peramine-coding genes, all of which had a bias to end with a C nucleotide. In the seven genomes analyzed, codon usage was biased in highly expressed coding sequences (CDSs) with shorter length and higher GC content. Genes in the peramine-coding gene cluster had higher GC content at the third nucleotide position of the codon, and highly expressed genes had higher GC content at the second position. In orthologous peramine-coding CDSs, high expression level was not significantly correlated with CDS length and GC content. Analysis of selection pressure identified that the genes orthologous to peramine genes were under purifying selection. There were no differences in codon usage bias and selection pressure between peramine product genes and non-functional peramine product genes. Our results provide insights into understanding codon evolution in Epichloë species.

BACKGROUND Near-infrared reflectance spectroscopy ( NIRS) has been widely used in forage quality control because it is faster, cleaner and less expensive than conventional chemical procedures. In Lolium perenne (perennial ryegrass), one of the most important forage grasses, the infection by asymptomatic Epichloë fungal endophytes alters the plant nutritional quality due to the production of alkaloids. In this research, we developed a rapid method based on NIRS to detect and quantify endophyte alkaloids (peramine, lolitrem B and ergovaline) using a heterogeneous set of L. perenne plants obtained from wild grasslands and cultivars. RESULTS NIR spectra from dried grass samples were recorded and classified according to the absence or presence of alkaloids, based on reference methods. The best discriminant equations for detection of alkaloids classified correctly 94.4%, 87.5% and 92.9% of plants containing peramine, lolitrem B and ergovaline, respectively. The quantitative NIR equations obtained by modified partial least squares (MPLS) algorithm had coefficients of correlation of 0.93, 0.41, and 0.76 for peramine, lolitrem B and ergovaline respectively. CONCLUSION NIRS is a suitable tool for qualitative analysis of endophyte alkaloids in grasses and for the accurate quantification of peramine and ergovaline. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Two novel regiocontrolled syntheses of pyrrolopyrazinones were developed. N -Methylpyrrole amide and 1-bromo-1-alkyne were annulated in the presence of a copper catalyst to give 3-substituted pyrrolopyrazinone in a regioselective manner. In contrast, heating N -methylpyrrole amide with the same haloalkyne in the presence of K 3 PO 4 provided the haloaminal, which was transformed regioselectively into 4-substituted pyrrolopyrazinone. The former procedure was successfully applied to the synthesis of peramine, a natural product isolated from an endophytic fungus. [ABSTRACT FROM AUTHOR]

In New Zealand, the fungal endophyte Epichloë festucae var. lolii Latch, Christensen & Samuels ( Hypocreales) is widely used to enhance productivity and survival of mature perennial ryegrass, Lolium perenne L. ( Poaceae) plants. These endophytes produce alkaloids that reduce damage by insect pests, such as Argentine stem weevil ( ASW), Listronotus bonariensis ( Kuschel) ( Coleoptera: Curculionidae). However, knowledge of the effect of these fungi on seedlings is limited. High concentrations of grass-endophyte-derived alkaloids occur in endophyte-infected seed and can be translocated into seedlings to provide early protection from insect herbivory. A glasshouse experiment was conducted in which adult ASW were given a choice between perennial ryegrass cultivar 'One50' without endophyte (Nil) or containing endophyte strains AR1, AR37, or NZCT, and cv. 'Trojan' containing strain NEA2, in three post-sowing time periods. ASW feeding damage was assessed together with plant survival and dry weight production. Plant survival and dry matter production was reduced in all treatments with mortality highest in endophyte-free and AR37 treatments, especially in the first and second time period. Strongest adult ASW deterrence occurred on seedlings infected with endophyte strains that produce peramine ( AR1, NZCT, and NEA2). Strain NZCT was virtually undamaged in the third time period whereas damage initially increased in AR1 and NEA2 up until day 50-53 post-sowing and then declined. Endophyte-free and AR37 treatments were significantly more damaged than AR1, NZCT, and NEA2. This research indicated there is a period of seedling vulnerability to adult ASW attack during germination, even for strains producing peramine. Possible explanations for these results are discussed. [ABSTRACT FROM AUTHOR]

Symbiotic associations between tall fescue grasses and asexual Epichloë fungal endophytes exhibit biosynthesis of alkaloid compounds causing both beneficial and detrimental effects. Candidate novel endophytes with favourable chemotypic profiles have been identified in germplasm collections by screening for genetic diversity, followed by metabolite profile analysis in endogenous genetic backgrounds. A subset of candidates was subjected to genome survey sequencing to detect the presence or absence and structural status of known genes for biosynthesis of the major alkaloid classes. The capacity to produce specific metabolites was directly predictable from metabolic data. In addition, study of duplicated gene structure in heteroploid genomic constitutions provided further evidence for the origin of such endophytes. Selected strains were inoculated into meristem-derived callus cultures from specific tall fescue genotypes to perform isogenic comparisons of alkaloid profile in different host backgrounds, revealing evidence for host-specific quantitative control of metabolite production, consistent with previous studies. Certain strains were capable of both inoculation and formation of longer-term associations with a nonhost species, perennial ryegrass ( Lolium perenne L.). Discovery and primary characterisation of novel endophytes by DNA analysis, followed by confirmatory metabolic studies, offers improvements of speed and efficiency and hence accelerated deployment in pasture grass improvement programs. [ABSTRACT FROM AUTHOR]

Screening of a variety of ligands and reaction conditions for the copper-catalyzed cross-coupling of alkynyl bromides with pyrroles, reveals that the use of the phenanthroline ligand 4,7-dimethoxy-1,10-phenanthroline affords a range of ynpyrroles in good to moderate yields. Furthermore, the utility of these ynpyrroles is demonstrated in the preparation of a series of pyrrolo[2,1-c][1,4]oxazin-1-ones and a formal total synthesis of the pyrrole natural product peramine. [ABSTRACT FROM AUTHOR]

The endophyte presence and diversity in natural populations ofPoa alsodeswere evaluated along a latitudinal transect from the southern distribution range in North Carolina to New York. Two distinctEpichloëhybrid taxa were identified from 23 populations. Each taxon could easily be distinguished by polymerase chain reaction (PCR) genotyping with primers designed to mating type genes and alkaloid biosynthesis genes that encode key pathway steps for ergot alkaloids, indole-diterpenes, lolines, and peramine. The most commonly foundEpichloëtaxon,Poa alsodesTaxonomicGroup-1(PalTG-1), was detected in 22 populations at high infection frequencies (72–100%), with the exception of one population at high elevation (26% infection). The second taxon, PalTG-2, was observed only in five populations in Pennsylvania constituting 12% of infected samples. Phylogenetic analyses placed PalTG-1 as an interspecific hybrid ofE. amarillansandE. typhinasubsp.poaeancestors, and it is considered a new hybrid species, which the authors nameEpichloë alsodes. PalTG-2 is an intraspecific hybrid of twoE. typhinasubsp.poaeancestors, similar toE. schardliifrom the hostCinna arundinacea, which the authors propose as a new variety,Epichloë schardliivar.pennsylvanica. Epichloë alsodesisolates were all mating typeMTA MTBand tested positive fordmaW, easC, perA, and someLOLgenes, but only the alkaloidN-acetylnorloline was detected inE. alsodes–infected plant material.Epichloë schardliivar.pennsylvanicaisolates were all mating typeMTB MTBand tested positive forperA, but peramine was not produced. BothE. alsodesandE. schardliivar.pennsylvanicaappeared to have completeperAgenes, but point mutations were identified inE. alsodesthat would render the encodedperAgene nonfunctional. [ABSTRACT FROM PUBLISHER]

The ecological effects of novel grass–endophyte associations used in agriculture have not been widely studied. Previous studies of asexual Epichloë- infected Lolium perenne suggest that endophyte concentration is altered in high sugar grasses (HSGs) selectively bred to produce higher concentrations of water-soluble carbohydrates relative to conventional cultivars. We investigated whether differences are due to the effects of altered carbohydrates, or genetic background, by growing multiple cultivars in both high-sugar trait expression and non-expression conditions (using light/temperature treatments). Endophyte and alkaloid concentrations were measured in three HSG and three NSG (normal-sugar grass) cultivars infected with Lp19 or AR37 endophyte strains. Low molecular weight (LMW) carbohydrates had a small effect, explaining <6% of the variation in endophyte concentration. Endophyte concentrations were strongly dependent on plant genotype and fungal strain, with the highest concentrations seen in Lp19, suggesting that the interaction is highly dependent on genetic compatibility. Changes in endophyte concentration due to altered environmental variables and genetic compatibility may have consequences for persistence, toxicity, and invasive potential of endophyte-infected plants. [ABSTRACT FROM AUTHOR]

Studying geographic variation of microbial mutualists, especially variation in traits related to benefits they provide their host, is critical for understanding how these associations impact key ecological processes. In this study, we investigate the phylogenetic population structure of Epichloë species within Bromus laevipes, a native cool-season bunchgrass found predominantly in California. Phylogenetic classification supported inference of three distinct Epichloë taxa, of which one was nonhybrid and two were interspecific hybrids. Inheritance of mating-type idiomorphs revealed that at least one of the hybrid species arose from independent hybridization events. We further investigated the geographic variation of endophyte-encoded alkaloid genes, which is often associated with key benefits of natural enemy protection for the host. Marker diversity at the ergot alkaloid, loline, indole-diterpene, and peramine loci revealed four alkaloid genotypes across the three identified Epichloë species. Predicted chemotypes were tested using endophyte-infected plant material that represented each endophyte genotype, and 11 of the 13 predicted alkaloids were confirmed. This multifaceted approach combining phylogenetic, genotypic, and chemotypic analyses allowed us to reconstruct the diverse evolutionary histories of Epichloë species present within B. laevipes and highlight the complex and dynamic processes underlying these grass-endophyte symbioses. [ABSTRACT FROM AUTHOR]

Plants form mutualistic symbioses with a variety of microorganisms including endophytic fungi that live inside the plant and cause no overt symptoms of infection. Some endophytic fungi form defensive mutualisms based on the production of bioactive metabolites that protect the plant from herbivores in exchange for a protected niche and nutrition from the host plant. Key elements of these symbioses are vertical transmission of the fungus through seed of the host plant, a narrow host range, and production of bioactive metabolites by the fungus., Grasses frequently form symbioses with endophytic fungi belonging to the family Clavicipitaceae. These symbioses have been studied extensively because of their significant impacts on insect and mammalian herbivores. Many of the impacts are likely due to the production of four classes of bioactive alkaloids - ergot alkaloids, lolines, indole-diterpenes and peramine - that are distributed in different combinations among endophyte taxa., Several legumes, including locoweeds, are associated with a toxic syndrome called locoism as a result of their accumulation of swainsonine. Species in two genera were recently found to contain previously undescribed endophytic fungi ( Undifilum spp., family Pleosporaceae) that are the source of that toxin. The fungi are strictly vertically transmitted and have narrow host ranges., Some plant species in the morning glory family ( Convolvulaceae) also form symbioses with endophytic fungi of the Clavicipitaceae that produce ergot alkaloids and, perhaps in at least one case, lolines. Other species in this plant family form symbioses with undescribed fungi that produce swainsonine. The swainsonine-producing endophytes associated with the Convolvulaceae are distinct from the Undifilum spp. associated with locoweeds and the Clavicipitaceous fungi associated with Convolvulaceae., In the establishment of vertically transmitted symbioses, fungi must have entered the symbiosis with traits that were immediately useful to the plant. Bioactive metabolites are likely candidates for such pre-adapted traits which were likely useful to the free-living fungi as well. With future research, vertically transmitted fungi from diverse clades with narrow host ranges and that produce bioactive compounds are likely to be found as important mutualists in additional plants. [ABSTRACT FROM AUTHOR]

Atmospheric CO2 is expected to increase to between 550 ppm and 1000 ppm in the next century. CO2-induced changes in plant physiology can have ecosystem-wide implications and may alter plant-plant, plant-herbivore and plant-symbiont interactions. We examined the effects of three concentrations of CO2 (390, 800 and 1000 ppm) and two concentrations of nitrogen fertilizer (0.004 g N/week versus 0.2 g N/week) on the physiological response of Neotyphodium fungal endophyte-infected and uninfected tall fescue plants. We used quantitative PCR to estimate the concentration of endophyte under altered CO2 and N conditions. We found that elevated CO2 increased the concentration of water-soluble carbohydrates and decreased the concentration of plant total amino acids in plants. Fungal-derived alkaloids decreased in response to elevated CO2 and increased in response to nitrogen fertilization. Endophyte concentration (expressed as the number of copies of an endophyte-specific gene per total genomic DNA) increased under elevated CO2 and nitrogen fertilization. The correlation between endophyte concentration and alkaloid production observed at ambient conditions was not observed under elevated CO2. These results suggest that nutrient exchange dynamics important for maintaining the symbiotic relationship between fungal endophytes and their grass hosts may be altered by changes in environmental variables such as CO2 and nitrogen fertilization. [ABSTRACT FROM AUTHOR]

Endophytic fungi in cool-season grass species produce herbivore-toxic alkaloids, which are assumed to harm higher trophic levels along food chains. Previous studies have shown fitness disadvantages for higher trophic levels that feed on aphids that were exclusively reared on perennial ryegrass ( Lolium perenne) infected with the endophytic fungus Neotyphodium lolii. However, it is unknown whether the alkaloids produced by the fungus-grass association can be assimilated by plant sap-sucking insects like aphids. Using an ultra high performance liquid chromatography method combined with mass spectrometry, we provide the first evidence that the alkaloids peramine and lolitrem B are present in aphids ( Rhopalosiphum padi) and in aphid predators when the aphids are reared on endophyte-infected grass. We conclude that alkaloids can enter the plant sap of the grass and are responsible for longer pupal stages of the ladybird Harmonia axyridis and for fitness disadvantages of aphids and their predators as shown in previous studies. [ABSTRACT FROM AUTHOR]

The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed. [ABSTRACT FROM AUTHOR]

To improve Neotyphodium endophyte-mediated resistance to black cutworm Agrotis ipsilon (Hufnagel) (BCW), a series of experiments was conducted by using several different cultivars of tall fescue, Schedonorus arundinaceus (Schreb.) Dumort. in combination with several different haplotypes of the endophyte Neotyphodium coenophialum (Morgan-Jones & Cams) (plant cultivar X endophyte haplotype = plant line), each producing unique alkaloid profiles. BCW settling response, survival at 5 and 10 d, and larval biomass varied significantly among plant lines. In general, greater variation BCW performance was observed within a single plant cultivar infected with different endophyte haplotypes than among different plant cultivars infected with the same endophyte haplotype, but comparisons among the former were far more numerous. Although five endophytemediated alkaloids representing three alkaloid classes were quantified in the plants, the pyrrolizidine alkaloid N-acetyl norloline was consistently the single best predictor of BCW performance. BCW settling response, 5-d survival, and 10-d survival decreased as levels of the alkaloid N-acetyl norloline increased. The same three response variables also decreased with increasing levels of peramine, but increased with increasing levels of ergovaline. Minor variation in endophyte infection levels occurring among infected plant lines bad no significant influence on BCW performance. Results indicate a potentially important role for N-acetyl norloline and peramine in providing resistance to black cutworm whereas ergovaline appears to be much less important. Therefore, endophyte haplotypes expressing high levels of N-acetyl norloline and peramine may be of particular importance for developing 'friendly' endophyte-enhanced turf and pasture grasses that resist challenging lepidopteran pests, although remaining safe for wildlife and grazing mammals. [ABSTRACT FROM AUTHOR]

Members of genus Neotyphodium are asexual derivatives of sexual Epichloë species and maintain endophytic relationships with many cool-season grasses. Most Neotyphodium species analyzed so far are interspecific hybrids with combined or partial genomes of two or three ancestral species. In this study we characterized Neotyphodium isolates from Cinna arundinacea, a perennial cool-season grass from eastern North America. A total of 23 isolates grouping into two distinct morphotypes were obtained from five local populations of C. arundinacea. PCR amplification and cloning of translation-elongation factor 1-α (tefA) and β-tubulin (tubB) genes of 10 isolates comprising both morphotypes (two isolates per location) revealed that all 10 contain two copies of tefA and tubB genes. Surprisingly phylogenetic analysis of mainly non-coding sequence from these genes revealed that both copies in each isolate were inherited from Epichloë typhina ancestors, indicating that the C. arundinacea endophytes arose through intraspecific hybridization between two E. typhina progenitors with extant relatives infecting hosts Poa nemoralis and Poa pratensis. Furthermore the tefA sequences were identical between isolates, as were tubB sequences, despite obvious morphological differences. Profiling of alkaloid biosynthetic genes from these isolates indicated the presence of the peramine biosynthetic gene (perA) and the absence of genes required for biosynthesis of lolines, indole-diterpenes and ergot alkaloids. Thus this endophyte is potentially capable of producing peramine in planta and providing protection to its host from insect pests. The absence of genes for indole-diterpenes and ergot alkaloid biosynthesis makes this endophyte a candidate for agricultural applications. Based on our phylogenetic analysis, alkaloid profiling and description of morphological characteristics, we propose the name Neotyphodium schardlii for these isolates from C. arundinacea, a new member of genus Neotyphodium and the first described to have arisen through intraspecific hybridization. [ABSTRACT FROM AUTHOR]

Peramine, a heterocyclic natural molecule, reveals two main, different in nature, electronic absorption bands. Theoretical calculations at the TD-B3LYP/6–311++G(d,p) level of theory show that the electronic excitations are connected predominantly with π → π* and charge-transfer (CT) transitions. Excitation of electrons from the pyrrolopyrazinone ring to the side chain plays a role in creating the CT transition. The character and energy of the first 30 singlet–singlet electronic transitions have also been investigated for the most stable conformation of peramine. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: The comparative analysis of IR and Raman spectra of peramine and its four derivatives in solid state was carried out. The harmonic vibrational frequencies, infrared intensities, and Raman scattering activities were calculated at density functional B3LYP methods with 6-311++G(d,p) basis set. For the predicted spectra, a potential energy distribution of normal modes was also calculated. For peramine derivatives the conjugation effect of pyrrole with pyrazinone ring was observed as a result of introduction of double bond. Moreover, 1H NMR analysis indicated that pyrrole protons are deshielded in comparison with the pyrrolopyrazinone model ring system. [Copyright &y& Elsevier]

Hordeum vulgare is an important source of feed and forage for livestock, and of food and drink for humans, but its utilization rate is lower than that of other cereal crops, thus it is crucial to improve barley agronomic traits and production. Epichloë bromicola is an endophyte that was isolated from wild barley (Hordeum brevisubulatum). Previous studies have found that Epichloë can indirectly influence the growth of host plants by affecting soil chemical characteristics, the microbial community, and by producing a range of secondary metabolites. However, underlying effects of Epichloë on the abundance and diversity of soil and root microbes have not been well-studied. In addition, there is a question regarding the relationship between endophyte-produced alkaloids and effects on the root and rhizosphere microbial communities. The objective of this study was to investigate changes in agronomic traits, nutritional properties, peramine, soil chemical and microbial community in the fourth generation of new barley symbionts EI (E. bromicola-infection) and EF (E. bromicola-free) in LQ+4 and LZ+4. We understand the plant height and biomass of EI in LZ+4 were significantly higher than those of EF. The HPLC analysis showed that the peramine content of EI in LQ+4 and LZ+4 was 0.085 and 0.1 mg/g, respectively. We compared the bacterial and fungal communities by analyzing the 16s rRNA (for bacteria) and ITS rDNA regions (for fungi). Our data revealed that the composition of fungal communities in rhizosphere soil of LZ+4 EI are higher than EF. In addition, the diversity and richness of fungal communities in root and rhizosphere soil of LQ+4 EI and LZ+4 EI are significantly higher than EF. Rhizosphere soil microbial community composition was higher than that in roots in LQ+4 and LZ+4. Peramine was significantly and positively correlated with the richness of the soil fungal community. Moreover, the principal component analysis (PCoA) results indicated that E. bromicola significantly influenced the community composition of root and rhizosphere soil microbes in both LQ+4 and LZ+4. Our results illustrate that E. bromicola can influence barley growth, peramine production and microbial communities associated with barley. [ABSTRACT FROM AUTHOR]

A series of laboratory experiments was carried out with the main objective of contributing to the development of biosafety testing methods for transgenic plants or plants produced by other novel technologies. The adult stage of a New Zealand native weevil, Nicaeana cervina (Broun) (Coleoptera: Curculionidae: Entiminae), was used as a test insect as this has become well-adapted to introduced pasture species, feeding on both ryegrass [ Lolium perenne L. (Poaceae)] and white clover [ Trifolium repens L. (Fabaceae)]. Ryegrass plants used in experiments were infected with strains of the symbiotic endophytic fungi Neotyphodium lolii (Latch, M.J. Chr. & Samuels) and Epichloe festucae Leuchtm., Schardl & M.R. Siegel and included a transgenic endophyte, which was modified to eliminate production of the insect deterrent, peramine. Nicaeana cervina was exposed to these endophyte-infected plants along with endophyte-free ryegrass. Weevils were also exposed to white clover with high or low levels of cyanogens, and to white clover selections bred conventionally to contain high or low levels of crude protein. These bioassays were carried out sometimes over a series of time intervals, and weevil survival, feeding, and reproductive performance variables were measured. Nicaeana cervina survival was generally high, although feeding and oviposition were variable between experiments despite high replication in some cases. A summary of significant differences obtained across all experiments suggested that data on the proportion of weevils feeding and amounts of leaf material consumed were more often significantly different than treatment effects on survival or reproductive performance. A positive relationship between the proportion of weevils feeding and those ovipositing was found, suggesting that a 50% reduction in feeding would result in a 28% reduction in the proportion of weevils ovipositing. Power analysis using the range of values recorded for the variables measured indicated that for proportions of weevils surviving, feeding, or fecund, about 35–42 replicates would be required for a 40% difference in values. For measures of leaf consumption and numbers of eggs per female, fewer replicates were required for 95% power when these values were at the higher end of the range than when they were at the lower end of the observed range. The usefulness of the case study test insect and risk assessment protocols used for biosafety testing are discussed. [ABSTRACT FROM AUTHOR]

Festuca rubra subsp. pruinosa is a grass that grows on coastal cliffs along the Atlantic coast of Europe. Asymptomatic plants of this species are systemically infected by the fungal endophyte Epichloë festucae. It is not known whether the alkaloids, ergovaline and peramine, are produced by the endophyte in F. rubra subsp. pruinosa. Plants from four populations were collected from the northern coast of Galicia (Spain) and examined for the presence of fungal endophytes. Ergovaline and peramine concentrations were analysed over two consecutive years, at two plant growth stages, and in different types of plant tissues. Infected plants of F. rubra subsp. pruinosa contained ergovaline but not peramine. Ergovaline was detected in 0·80 of the plants, with concentrations ranging from 0·05 to 1·9 μg g−1 dry matter. The differences in ergovaline concentration between different types of plant tissues (vegetative and reproductive), plant populations and sampling dates were not statistically significant. [ABSTRACT FROM AUTHOR]

• The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. • Here, we infected two perennial ryegrass ( Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. • Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. • The results stress the need for wider quantification of fungal endophytes in the grassland–foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems. [ABSTRACT FROM AUTHOR]

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]

Abstract: The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid–endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline–endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B–endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed. [Copyright &y& Elsevier]

Summary: • Variability in the fungal endophytes of 83 natural populations of Lolium perenne (perennial ryegrass) from Europe was assessed.• One plant per population was used for endophyte isolation and mycotoxin analysis. Variability in three isozymes, colony morphology and growth rate on potato dextrose agar (PDA), and synthesis of ergovaline, lolitrem B and peramine was recorded.• Three species were found among 94 strains isolated: Neotyphodium lolii, Neotyphodium sp. (LpTG‐2) and Gliocladium‐like. The most frequent species was N. lolii, which showed high variability. In 12 populations, a single plant harboured two different endophytes. One‐third of the isolates of N. lolii did not produce ergovaline whereas a few isolates did not produce lolitrem B. Ergovaline and lolitrem B‐deficient strains, but not the few peramine‐deficient isolates, had characteristic morphologies on PDA. No isolate was deficient for both ergovaline and lolitrem B synthesis.• Selection of ergovaline and lolitrem‐deficient strains based only on the morphology of the isolates in culture may be possible. [ABSTRACT FROM AUTHOR]

The three alkaloid groups—lolines, ergopeptides, and peramine— are typically associated with endophyte infection of grasses, with the main function to protect hosts against herbivores. We determined levels of N-formylloline, N-acetylloline, ergovaline, and peramine in 18 European grasses naturally infected with seed-transmitted Neotyphodium endophytes or sexual Epichloë species. Peramine was the most common alkaloid, whereas lolines and ergovaline were only detected in Festuca hosts infected with E. festucae, N. coenophialum, or N. uncinatum. Only ten of the grass species analyzed contained detectable amounts of one or more of these alkaloids. There was a clear tendency for plants associated with stroma-forming Epichloë species to be free of alkaloids, and those that did produce alkaloids contained only small levels of peramine. In contrast, plants infected with seed-transmitted Neotyphodium endophytes often contained extremely high levels of lolines. Lolines enhance host survival through increased protection from herbivores and, thus, may be particularly favored in asexual endophytes that depend on host seed-production for their dispersal. [ABSTRACT FROM AUTHOR]

Four Chewings fescue and two strong creeping red fescue selections that had been artificially inoculated and stably maintained with four different endophytes were evaluated in feeding trials with chinch bugs ( Blissus leucopterus hirtus). Significant differences in survival were found between the endophyte-inoculated plants and their endophyte-free counterparts. After seven days, 54.2% of chinch bugs were alive on endophyte-free tillers versus only 7.4% of chinch bugs fed tillers from endophyte-inoculated plants. Some differences were also found among the various plant–endophyte combinations. In Petri dish preference trials, chinch bugs showed a preference for the CA endophyte (obtained from a Chewings fescue) over the RC endophyte (obtained from a strong creeping red fescue) in Chewings fescue selection C1117. Only the inoculated plants produced erogvaline, peramine, and lolitrem B; moreover, significant differences were found among the plant–endophyte combinations in the levels of these alkaloids. The Chewings selections C1117 and C1090 produced more ergovaline, and C1090 more lolitrem B, than other plants, regardless of endophyte source. In the presence of the RC endophyte, more ergovaline and lolitrem B was produced than in the presence of the CA endophyte regardless of plant genotype. Both host plant and endophyte, therefore, contributed factors that determined alkaloid production. No significant correlations between chinch bug survival and alkaloid levels were found, however, and overall, no one plant genotype or endophyte source proved to be significantly more toxic than another to chinch bugs. Nevertheless, the results clearly demonstrated that artificial inoculations of endophyte-free fescue genotypes can produce plants with increased toxicity to chinch bugs. [ABSTRACT FROM AUTHOR]

Through the production of alkaloids and mycotoxins, the endophytic fungus of perennial ryegrass, Neotyphodium lolii, confers resistance to the host grass against several insect pasture pests and causes health problems in livestock. The effects of plant component, plant reproductive development, and plant management (trimming) on the concentration and accumulation of peramine, the alkaloid thought to be most important for conferring resistance against Argentine stem weevil, were investigated. Clones of eight different naturally occurring perennial ryegrass- N. lolii associations were used in the experiment, which was conducted during spring–summer, which corresponded with the height of plant reproductive activity. Clones were either trimmed regularly to maintain them in a vegetative state (V clones) or not trimmed, allowing them to become fully reproductive (R clones). V and R clones of each grass/endophyte association were destructively harvested at times that corresponded roughly with the start, middle, and end of the plant reproductive phase. Harvested material was dissected into a number of plant components that were analyzed for peramine concentration by HPLC. All clippings from the V clones were also analyzed. Peramine concentrations were usually similar in leaf sheaths and leaf blades, but they decreased as leaf age increased. The seed from R clones and younger sheaths and blades of leaves from vegetative tillers contained the highest concentrations, while the root, crown, and dead leaf tissue contained the lowest. During the inflorescence phase, tissues from reproductive tillers generally contained lower concentrations than corresponding tissues from vegetative tillers. Also at this time, mean peramine concentrations were higher in V clones than in R clones. By the end of the plant reproductive phase, at the mature seed stage, mean peramine concentrations in R and V clones were similar. The seed component, which contained approximately 75% of the total peramine present in the plant (at a mean concentration of 63.7 μg/g), probably accounted for much of the recovery in the R clones. [ABSTRACT FROM AUTHOR]