Feeding Performance of Argentine Stem Weevil Is Reduced by Peramine from Perennial Ryegrass Infected with Endophyte Fungus.

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.

Identification of Three Epichloë Endophytes from Hordeum bogdanii Wilensky in China.

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.

Sub-acute feeding study of a tall fescue endophyte in a perennial ryegrass host using mice.

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.

Inoculation of Barley (Hordeum vulgare) with the Endophyte Epichloë bromicola Affects Plant Growth, and the Microbial Community in Roots and Rhizosphere Soil.

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.

A Tale of Two Grass Species: Temperature Affects the Symbiosis of a Mutualistic Epichloë Endophyte in Both Tall Fescue and Perennial Ryegrass.

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.

Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis.

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.

A concise synthesis of peramine, a metabolite of endophytic fungi.

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.

Comprehensive Analysis of Codon Usage Bias in Seven Epichloë Species and Their Peramine-Coding Genes.

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.

Interspecific and intraspecific hybrid Epichloë species symbiotic with the North American native grass Poa alsodes.

The endophyte presence and diversity in natural populations of Poa alsodes were evaluated along a latitudinal transect from the southern distribution range in North Carolina to New York. Two distinct Epichloë hybrid 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 found Epichloë taxon, Poa alsodes Taxonomic Group-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 of E. amarillans and E. typhina subsp. poae ancestors, and it is considered a new hybrid species, which the authors name Epichloë alsodes. PalTG-2 is an intraspecific hybrid of two E. typhina subsp. poae ancestors, similar to E. schardlii from the host Cinna arundinacea, which the authors propose as a new variety, Epichloë schardlii var. pennsylvanica. Epichloë alsodes isolates were all mating type MTA MTB and tested positive for dmaW, easC, perA, and some LOL genes, but only the alkaloid N-acetylnorloline was detected in E. alsodes-infected plant material. Epichloë schardlii var. pennsylvanica isolates were all mating type MTB MTB and tested positive for perA, but peramine was not produced. Both E. alsodes and E. schardlii var. pennsylvanica appeared to have complete perA genes, but point mutations were identified in E. alsodes that would render the encoded perA gene nonfunctional.

Qualitative and quantitative analysis of endophyte alkaloids in perennial ryegrass using near-infrared spectroscopy.

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.

Genomic and metabolic characterisation of alkaloid biosynthesis by asexual Epichloë fungal endophytes of tall fescue pasture grasses.

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.

Interspecific hybridization and bioactive alkaloid variation increases diversity in endophytic Epichloë species of Bromus laevipes.

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.

Metabolite analysis of the effects of elevated CO2 and nitrogen fertilization on the association between tall fescue (Schedonorus arundinaceus) and its fungal symbiont Neotyphodium coenophialum.

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.

The relationship between mycotoxin synthesis and isolate morphology in fungal endophytes of Lolium perenne.

• 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.