Pathological changes seen in horses in New Zealand grazing Mediterranean tall fescue (Lolium arundinaceum) infected with selected endophytes (Epichloë coenophiala) causing equine fescue oedema.

AIM: To investigate whether Mediterranean tall fescue (Lolium arundinaceum (Schreb.) Darbysh. (syn Festuca arundinacea)) infected with selected fungal endophytes (Epichloë coenophiala (formerly Neotyphodium coenophialum)) caused equine fescue oedema when grown in New Zealand, and to examine the pathological changes associated with this intoxication. METHODS: Horses were grazed on Mediterranean tall fescue that was infected with the endophytes AR542 (n=2), or AR584 (n=3), or Mediterranean tall fescue that was endophyte-free (n=2). Blood samples were taken up to 7 days after the start of feeding to detect changes in concentrations of total protein in serum and packed cell volume. Any horse showing clinical evidence of disease was subject to euthanasia and necropsy. RESULTS: Within 6 days, both horses grazing fescue infected with AR542 became depressed and lethargic. One horse grazing fescue infected with endophyte AR584 became depressed within a 5-day feeding period while another horse in this group died shortly after being removed from the AR584 pasture. The third horse in this group did not develop clinical signs within the 5-day feeding period. However, haemoconcentration and hypoproteinaemia was detected in all horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 endophyte. No abnormalities were observed in horses grazing fescue that was endophyte-free. Necropsy examination was performed on two horses grazing fescue infected with AR542 and one horse grazing fescue infected with AR584. All three horses had marked oedema of the gastrointestinal tract. Histologically, the oedema was accompanied by large numbers of eosinophils, but no necrosis. CONCLUSIONS: Horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 developed hypoproteinaemia and haemoconcentration, most likely due to leakage of plasma proteins into the gastrointestinal tract. This suggests that these selected endophytes produce a compound that is toxic to horses, although the toxic principle is currently unknown. CLINICAL RELEVANCE: Mediterranean tall fescue that is infected by AR542 or AR584 should not be fed to horses in New Zealand. This intoxication should be considered in horses in which a rapid onset of hypoproteinaemia and haemoconcentration is detected. This intoxication should also be considered if marked gastrointestinal oedema is observed.

Neotyphodium Endophyte Changes Phytoextraction of Zinc in Festuca arundinacea and Lolium perenne.

The effect of Neotyphodium endophytes on growth parameters and zinc (Zn) tolerance and uptake was studied in two grass species of Festuca arundinacea and Lolium perenne. Plants were grown under different Zn concentrations (control, 200, 400, 800, and 1800 mg kg(-1)) in potted soil for 5 months. The results showed that the number of plant tillers was 85 and 51% greater in endophyte infected Festuca (FaEI) and Lolium (LpEI), respectively, compared to their endophyte free (EF) plants. Roots and shoots dry weights in infected Festuca were 87 and 9% greater than non-infected counterparts but in opposite, EF Lolium had 47 and 8% greater root and shoot dry weights than LpEI. Endophyte infected Festuca and Lolium improved chlorophyll fluorescence as Fv/Fm at high concentrations of Zn, showing their better chlorophyll functions and significant reduction of Zn stress in endophyte infected plants. Shoots of endophyte infectedFestuca had 82% greater concentration of Zn than EF Festuca when grown in soil containing 1800 mg kg(-1) Zn. Festuca and Lolium may tolerate high Zn concentration in soil without reduction in shoot and root growth. Endophyte infection in Festuca may help the grass accumulate and transport more Zn in aboveground parts under Zn-stress, thereby aiding phytoremediation of contaminated soils.

Evidence of dehydration and electrolyte disturbances in cases of perennial ryegrass toxicosis in Australian sheep.

CASE REPORT: Perennial ryegrass toxicosis (PRGT) is a common disease entity in Australia, presenting as an association of clinical signs including alterations in normal behavioural, ataxia ('staggers'), ill thrift and gastrointestinal dysfunction ('scours'). Clinical signs can range in severity from mild (gait abnormalities and failure to thrive) to severe (seizures, lateral recumbency and death). Presentation across the flock is usually highly variable. PRGT is caused by toxins produced by the endophytic fungus Neotyphodium lolii, a symbiont of perennial ryegrass that is present in pastures across the temperate regions of Australia and Tasmania. A particular feature of PRGT in Australia is the occasional occurrence of large-scale sheep losses, suggesting other factors are influencing mortality rates compared with other PRGT risk zones such as North America and New Zealand. During 2011, producers in the state of Victoria experienced a mild outbreak of PRGT that affected large numbers of animals but with limited mortalities. Clinical samples taken from affected sheep showed a high incidence of dehydration and electrolyte abnormalities. CONCLUSION: We speculate that changes in hydration status may be a contributory aetiological factor in those years in which high numbers of deaths are associated with PRGT outbreaks in Australia.

Loline alkaloid production by fungal endophytes of Fescue species select for particular epiphytic bacterial microflora.

The leaves of fescue grasses are protected from herbivores by the production of loline alkaloids by the mutualist fungal endophytes Neotyphodium sp. or Epichloë sp. Most bacteria that reside on the leaf surface of such grasses can consume these defensive chemicals. Loline-consuming bacteria are rare on the leaves of other plant species. Several bacterial species including Burkholderia ambifaria recovered from tall fescue could use N-formyl loline as a sole carbon and nitrogen source in culture and achieved population sizes that were about eightfold higher when inoculated onto plants harboring loline-producing fungal endophytes than on plants lacking such endophytes or which were colonized by fungal variants incapable of loline production. In contrast, mutants of B. ambifaria and other bacterial species incapable of loline catabolism achieved similarly low population sizes on tall fescue colonized by loline-producing Neotyphodium sp. and on plants lacking this endophytic fungus. Lolines that are released onto the surface of plants benefiting from a fungal mutualism thus appear to be a major resource that can be exploited by epiphytic bacteria, thereby driving the establishment of a characteristic bacterial community on such plants.

Dynamics of Neotyphodium uncinatum and N-formylloline in Italian ryegrass, and their relation to insect resistance in the field.

AIMS: A fungal endophyte, Neotyphodium uncinatum, accumulates N-formylloline, which is toxic to Hemipteran insects, in Italian ryegrass. This study aimed to clarify the dynamics of N. uncinatum and N-formylloline in Italian ryegrass, and their relationship to insect resistance. METHODS AND RESULTS: Changes in the density and localization of N. uncinatum and N-formylloline in N. uncinatum-infected Italian ryegrass were examined by real-time PCR and gas chromatography, respectively. Neotyphodium uncinatum multiplied on pseudostems at the flowering stage, and then increased on inflorescences at the ripening stage. On the other hand, N-formylloline accumulated heavily in inflorescences and leaf blades, but lightly in pseudostems at the ripening stage. In field experiments, N. uncinatum-infected Italian ryegrass suppressed the occurrence of Stenotus rubrovittatus, which fed on the inflorescences, but was not effective to Laodelphax striatellus, which do not necessarily prefer a particular plant tissue. CONCLUSION: Localization of N. uncinatum and N-formylloline were discordant in Italian ryegrass. The N. uncinatum-infected Italian ryegrass was effective to only insects that prefer to feed on particular plant tissues containing N-formylloline. SIGNIFICANCE AND IMPACT OF STUDY: Our data implies that the relationship between insect habits and the dynamics of alkaloids in plants is important for the effective use of endophyte-infected crops.

Peramine and lolitrem B from endophyte-grass associations cascade up the food chain.

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.

Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae.

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.

Efficacy of domperidone gel in an induced model of fescue toxicosis in periparturient mares.

The objective was to evaluate the efficacy of domperidone in the prevention of reproductive complications of fescue toxicosis in periparturient mares. Pregnant mares at ≤310 days of gestation were fed ≥200 µg ergovaline per kg diet daily in endophyte-infected fescue hay and seed, starting ≥30 days before their expected foaling date (EFD: 340 days after breeding). Thirty-five mares were randomized to a treatment group to receive either domperidone gel (n = 20, 1.1 mg/kg, PO, once daily) or placebo (n = 15). Treatment was initiated 10 to 15 days before the EFD and continued for 5 days after foaling. "Treatment success" was defined as foaling within 14 days of the EFD, adequate mammary development on the day of foaling, and adequate lactation for 5 days postpartum. Twenty-seven mares were included in the effectiveness analysis. More mares in the domperidone group (12/13, P < 0.0001) were treatment successes than in the control group (1/14). Gestation length was shorter (P = 0.0011), and lactation at foaling (P = 0.0011) was better for the domperidone-group mares. Foals from two control mares were born dead and four others died or were euthanized within a few days after birth, compared with one foal death (an autolyzed twin) from a domperidone-treated mare. Plasma IgG concentrations were evaluated in 24 foals. Failure of passive transfer of immunoglobulins (IgG <800 mg/dL) occurred in 13/16 (81%) foals of domperidone-group mares and 7/8 (88%) foals of control mares. In conclusion, the reproductive complications of fescue toxicosis in periparturient mares induced by a fescue seed/hay model were prevented by treatment with domperidone.

A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne.

The endophytic fungus Neotyphodium lolii forms symbiotic associations with perennial ryegrass (Lolium perenne) and infection is typically described as asymptomatic. Here we describe a naturally occurring New Zealand N. lolii isolate that can induce dwarfing of L. perenne and suppress floral meristem development in the dwarfed plants. Further to this we demonstrate that the observed host dwarfing correlates with a reversible morphological change in the endophyte that appears associated with colony age. Mycelium isolated from normally growing plants had a typical cottony appearance in culture whereas mycelium from dwarfed plants appeared mucoid. Cottony colonies could be induced to turn mucoid after prolonged incubation and seedlings inoculated with this mucoid mycelium formed dwarfed plants. Mucoid colonies on the other hand could be induced to form cottony colonies through additional further incubation and these did not induce dwarfing. The reversibility of colony morphology indicates that the mucoid dwarfing phenotype is not the result of mutation. Ten isolates from other locations in New Zealand could also undergo the reversible morphological changes in culture, induce dwarfing and had the same microsatellite genotype as the original isolate, indicating that a N. lolii genotype with the ability to dwarf host plants is common in New Zealand.

The evaluation of endophyte toxin residues in sheep fat.

AIM: To monitor changes in concentrations of lolitrem B and epoxy-janthitrems in the fat of sheep grazing perennial ryegrass infected with wild-type- and AR37-endophyte, respectively, during the time of year when ryegrass staggers would be expected to be observed. METHODS: Ten 5-month-old lambs with no previous exposure to endophytes were grazed on either wild-type (containing lolitrem B, n = 5) or AR37 (containing epoxy-janthitrems, n = 5) endophyte-infected perennial ryegrass (Lolium perenne L.) pastures between October 2008 and June 2009. Animals were regularly assessed for ryegrass staggers using the Keogh scale (0 = no signs, 5 = severe tremors). When a score of > 3.5 was observed animals were removed from the treatment pastures for 1 month. Fat biopsy samples were taken from each animal at approximately monthly intervals and analysed for endophyte metabolites using high performance liquid chromatography (HPLC) methods developed during this study. Regular herbage samples were also taken and concentrations of endophyte metabolites measured. RESULTS: Efficient and reproducible methods to analyse both lolitrem B and epoxy-janthitrems in fat were developed. Concentrations of lolitrem B and epoxy-janthitrems in herbage and in sheep fat increased from late November to peak in mid-February. Ryegrass staggers was observed in both groups of sheep at this time. Following 1 month of grazing non-infected pasture mean concentrations in fat of lolitrem B decreased by 43% from 61.8 to 35.3 ppb, and of epoxy-janthitrems by 38% from 1032.0 to 639.5 ppb. Maximum concentrations in herbage of epoxy-janthitrems (35.7 ppm) were higher than of lolitrem B (3.4 ppm), but signs of staggers were less severe in sheep grazing pasture containing the former compared with the latter (median Keogh scores in late February were 2 and 3, respectively), consistent with epoxy-janthitrems being low potency toxins. CONCLUSION: This study demonstrated that concentrations of epoxy-janthitrems and lolitrem B in sheep fat increased quickly during the initial phase of the study when concentrations in pasture increased, and decreased when animals were removed from pastures containing these compounds. These data will be used in the risk assessment of the endophyte metabolites.

The Cre/lox system: a practical tool to efficiently eliminate selectable markers in fungal endophytes.

Interspecific hybridization is a common evolutionary process for the many epichloid fungi that consequently possess multiple gene copies accumulated from their parental strains. Serial gene manipulations in such strains are impeded by the limited availability of selectable resistance marker genes. Therefore, we developed a method for marker elimination suitable for a range of filamentous fungi that allows the reuse of the same marker for successive manipulations, and can also generate gene knockout mutants free of any foreign genes. For epichloae, the complete elimination of the marker gene from the genome would mitigate public concerns and regulatory hurdles to the use of such fungal strains in field experiments.

Are loline alkaloid levels regulated in grass endophytes by gene expression or substrate availability?

Many cool-season grasses (Poaceae, subfam. Pooideae) possess seedborne fungal symbionts, the epichloae, known for their bioprotective properties, and especially for production of anti-insect alkaloids such as lolines. Asexual epichloae (Neotyphodium species) are primarily or entirely transmitted vertically, whereas the sexual structures (stromata) of the related Epichloë species give rise to horizontally transmissible spores (ascospores). In certain grass-Neotyphodium species symbiota, levels of lolines are extremely high and apparently limited by availability of precursor amino acids, whereas sexual epichloae generally produce much lower levels. This may reflect the inherent conflict between the vertical and horizontal transmission; although the plant and seeds may be protected by the alkaloids, the sexual cycle depends on anthomyiid flies for cross-fertilization. Given this insect role, we predicted that loline biosynthesis would be down-regulated in the stromata relative to the corresponding asymptomatic tissues (inflorescences) of the same symbiota. This prediction was substantiated, and RNA-seq and RT-qPCR analysis indicated that the loline biosynthesis genes are dramatically upregulated in asymptomatic inflorescences compared to stromata. The fundamental difference between asexual and sexual epichloae in regulation of loline alkaloid levels is in keeping with evolutionary trends for greater host control on metabolism of their vertically transmitted symbionts compared to contagious symbionts.

Asexual endophytes and associated alkaloids alter arthropod community structure and increase herbivore abundances on a native grass.

Despite their minute biomass, microbial symbionts of plants potentially alter herbivory, diversity and community structure. Infection of grasses by asexual endophytic fungi often decreases herbivore loads and alters arthropod diversity. However, most studies to date have involved agronomic grasses and often consider only infection status (infected vs. uninfected), without explicitly measuring endophyte-produced alkaloids, which vary among endophyte isolates and may impact consumers. We combined field experiments and population surveys to investigate how endophyte infection and associated alkaloids influence abundances, species richness, evenness and guild structure of arthropod communities on a native grass, Achnatherum robustum (sleepygrass). Surprisingly, we found that endophyte-produced alkaloids were associated with increased herbivore abundances and species richness. Our results suggest that, unlike what has been found in agronomic grass systems, high alkaloid levels in native grasses may not protect host grasses from arthropod herbivores, and may instead more negatively affect natural enemies of herbivores.

Contractile response of fescue-naive bovine lateral saphenous veins to increasing concentrations of tall fescue alkaloids.

Various alkaloids found in endophyte-infected tall fescue have been shown to elicit different effects in the grazing animal. As part of an ongoing characterization of vascular response generated by different alkaloids, the objective of this study was to examine the vasoconstrictive potentials of ergonovine (a simple lysergic acid derivative) and alpha-ergocryptine, ergocristine, and ergocornine (all ergopeptine alkaloids) using bovine lateral saphenous veins (cranial branch) biopsied from fescue-naïve cattle. Segments (2 to 3 cm) of vein were surgically biopsied from healthy crossbred yearling cattle (n = 18; 274 +/- 8 kg of BW). Veins were trimmed of excess fat and connective tissue, sliced into 2 to 3 mm sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O(2)/5% CO(2); pH = 7.4; 37 degrees C). Tissue was allowed to equilibrate at 1 g of tension for 90 min before initiation of treatment additions. Increasing doses of each alkaloid (1 x 10(-10) to 1 x 10(-4) M) were administered every 15 min after buffer replacement. Data were normalized as a percentage of contractile response induced by a reference dose of norepinephrine (1 x 10(-4) M). Exposure of vein segments to increasing concentrations of ergocryptine, ergocristine, and ergonovine did not result in a contractile response until 1 x 10(-7) M, and ergocornine was even less potent (P < 0.05). Ergonovine had a greater maximal contractile intensity than ergocristine and ergocryptine (P < 0.05), with the 1 x 10(-4) M responses of ergonovine, ergocristine, ergocryptine, and ergocornine reaching maximums of 68.5 +/- 4.1, 45.5 +/- 4.5, 42.9 +/- 4.1%, and 57.2 +/- 9.9% of the norepinephrine maximum, respectively. The contractile response to increasing concentrations of ergonovine vs. ergocryptine, ergocristine, and ergocornine were opposite from previous evaluations of ergoline (e.g., lysergic acid) and ergopeptine (e.g., ergovaline) alkaloids using this bioassay, where the ergopeptine generated the greater contractile intensity. These data indicate that ergopeptines structurally different only at a single position of the peptide moiety do not exhibit differing contractile responses when considering contractile intensity. This difference may alter the potency when considering ergocornine was less potent than ergocryptine or ergocristine. These alkaloids may need to be considered when evaluating causative agents vasoconstriction associated with tall fescue-induced toxicosis.

Variation in endophyte-plant associations influence black cutworm (Lepidoptera: Noctuidae) performance and susceptibility to the parasitic nematode Steinernema carpocapsae.

This study examined how variability in Neotyphodium endophyte-grass associations influences black cutworm Agrotis ipsilon Hufnagel performance and susceptibility to the entomopathogenic nematode Steinernema carpocapsae (Weiser). Second-instar cutworm larvae were confined to greenhouse pots containing four different tall fescue Schedonorus phoenix (Scop.) Holub cultivars. After 1 wk, larvae were recovered from the pots, weighed, and individually exposed to 20 infective juvenile nematodes. Nematode-induced mortality was monitored for 72 h after exposure. Endophyte infection levels and ergot alkaloid concentrations varied between tall fescue cultivars, but endophyte infection level was not a significant predictor of ergot alkaloid concentrations in above-ground plant tissue. Larval survival also varied between cultivars, but neither endophyte infection level nor ergot alkaloid concentration was a significant covariate. Larval susceptibility to the entomopathogenic nematode varied between cultivars at 48 and 72 h after exposure. In all but one cultivar (Plantation), cumulative mortality at 72 h decreased significantly as ergot alkaloid concentrations increased. Neither larval biomass nor endophyte infection levels in tall fescue were significant predictors of larval susceptibility to the nematode. Results show that variation in endophyte-plant associations can influence black cutworm performance and susceptibility to entomopathogenic nematodes and that susceptibility to the nematode H. bacteriophora may be partially tied to ergot alkaloid levels in the insects' food. Findings further support the assertion that black cutworm may use certain endophyte-mediated toxins, particularly ergot alkaloids, as a form of acquired chemical defense against nematode-induced septicaemia.

Chemotaxis disruption in Pratylenchus scribneri by tall fescue root extracts and alkaloids.

Tall fescue (Festuca arundinacea) forms a symbiotic relationship with the clavicipitalean fungal endophyte Neotyphodium coenophialum. Endophyte-infected grass is tolerant to nematode, but the factors responsible are unknown. One objective of this work was to determine if root extracts of tall fescue effected chemoreceptor activity of Pratylenchus scribneri by using an in vitro chemoreception bioassay. Another objective was to determine if specific ergot alkaloids (ergovaline, ergotamine, a-ergocryptine, ergonovine), and loline alkaloids, all produced by the fungal endophyte, altered chemotaxis with this bioassay. Methanolic extract from roots altered chemotaxis activities in this nematode but only from roots of plants cultured 45 > or = d, which repelled nematodes. Extracts prepared from noninfected grasses were attractants. This assay indicated that the alkaloids were either repellents or attractants. N-formylloline was an attractant at concentrations of 20 microg/ml and lower, while at higher concentrations it was a repellent. Ergovaline, the major ergot alkaloid produced by the endophyte, was repellent at both high and low concentrations and caused complete death of the nematodes.

Indole-diterpene biosynthetic capability of epichloë endophytes as predicted by ltm gene analysis.

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