Sarah C. Finch

The exploitation of epichloae endophytes for agricultural benefit

Epichloae endophytes of family Clavicipitaceae (comprising genera Epichloë and Neotyphodium) are fungal symbionts of Pooideae grasses. The associations formed, range from mutually beneficial to antagonistic and the nature of this relationship is dependent upon the importance of vertical (via host seeds) versus horizontal (ascospore mediated) transmission of the fungus. These endophytes can enhance their hosts’ survival through protection from abiotic and biotic stresses and can thus be utilized in an agricultural context. Animal-safe grass-endophyte associations that confer bio-protective properties for increased pasture persistence and productivity have been developed and commercialized. One of the crucial drivers underpinning the selection of epichloae strains for commercial development is endophyte derived bioactivity. The potential of next generation endophytes is determined by testing a number of attributes such as agronomic fitness, animal and food safety as well as compatibility with host plants of interest. Strategic research supports these activities by focusing on elucidating mechanisms of compatibility between host and fungal symbiont, as well as investigating other molecular drivers of symbiosis such as siderophore mediated iron-uptake, fungal signalling, fungal growth in host plants and fungal secondary metabolism. This review weaves together the different strands of multidisciplinary research aimed at ultimately exploiting epichloae endophytes for increased pasture performance.

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K+ Channels

“Ryegrass staggers” is a neurological condition of unknown mechanism that impairs motor function in livestock. It is caused by infection of perennial ryegrass pastures by an endophytic fungus that produces neurotoxins, predominantly the indole-diterpenoid compound lolitrem B. Animals grazing on such pastures develop uncontrollable tremors and become uncoordinated in their movement. Lolitrem B and the structurally related tremor inducer paxilline both act as potent large conductance calcium-activated potassium (BK) channel inhibitors. Using patch clamping, we show that their different apparent affinities correlate with their toxicity in vivo. To investigate whether the motor function deficits produced by lolitrem B and paxilline are due to inhibition of BK ion channels, their ability to induce tremor and ataxia in mice deficient in this ion channel (Kcnma1-/-) was examined. Our results show that mice lacking Kcnma1 are unaffected by these neurotoxins. Furthermore, doses of these substances known to be lethal to wild-type mice had no effect on Kcnma1-/- mice. These studies reveal the BK channel as the molecular target for the major components of the motor impairments induced by ryegrass neurotoxins. Unexpectedly, when the response to lolitrem B was examined in mice lacking the β4 BK channel accessory subunit (Kcnmb4-/-), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK β4 subunit in motor control. The β4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.

A review of the Neotyphodium lolii / Lolium perenne symbiosis and its associated effects on animal and plant health, with particular emphasis on ryegrass staggers

Abstract Ryegrass staggers is a seasonal mycotoxicosis of grazing livestock characterised by tremors, in coordination and a staggering gait almost unaccompanied by physical lesions. Deaths occur only as a consequence of accident or starvation. Outbreaks, in summer and autumn, occur only on pasture in which endophyte (Neotyphodium lolii)-infected perennial ryegrass (Lolium perenne) predominates and usually on which animals are grazed intensively. Animals recover when moved to a different type of grazing or after rain has promoted pasture growth. The disease was recognised for 80 years before its cause was discovered as a consequence of a grazing trial of sheep on three ryegrass cultivars which happened to have three different levels of endophyte infection. The endophyte was first formally described as Acremonium loliae, later corrected to Acremonium lolii, and was finally placed in the genus Neotyphodium. It produces a number of secondary metabolites of which lolitrem B is the principal one causing ryegrass staggers symptoms. Ergopeptides are also produced which cause heat stress and lack of productivity. N. lolii is symptomless in the plant, seed borne and grows intercellularly in the aerial parts, mainly in reproductive tillers and leaf sheaths but sparsely in leaf blades. It dies in stored seed and infection rates of different ryegrass cultivars have depended on seed storage times during their production. In addition, N. Lolii produces insect feeding deterrents, among them peramine, which protects infected plants from pest predation. Because of this, control of ryegrass staggers by elimination of endophyte-infected ryegrass is not feasible in areas in which insect predation is a serious pasture problem. However, N. lolii strains vary in the secondary metabolites they produce allowing the selection of strains that produce desirable metabolites. By inoculating such strains into uninfected ryegrass plants it is possible to produce cultivars which do not cause ryegrass staggers but resist insect predation. This review aims to provide a comprehensive summary of the current understanding of the N. lolii / L. perenne symbiosis, the toxins it is known to produce, their effects on animals and plants and the strategies used to control their ill effects while maximising their beneficial ones

A role for BK channels in heart rate regulation in rodents.

The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na+ and Ca2+ and outward K+ currents. There are a number of K+ channel types expressed in the heart that play key roles in regulating the cardiac cycle. Large conductance calcium-activated potassium (BK) ion channels are not thought to be directly involved in heart function. Here we present evidence that heart rate can be significantly reduced by inhibiting the activity of BK channels. Agents that specifically inhibit BK channel activity, including paxilline and lolitrem B, slowed heart rate in conscious wild-type mice by 30% and 42%, respectively. Heart rate of BK channel knock-out mice (Kcnma1−/−) was not affected by these BK channel inhibitors, suggesting that the changes to heart rate were specifically mediated through BK channels. The possibility that these effects were mediated through BK channels peripheral to the heart was ruled out with experiments using isolated, perfused rat hearts, which showed a significant reduction in heart rate when treated with the BK channel inhibitors paxilline (1 µM), lolitrem B (1 µM), and iberiotoxin (0.23 µM), of 34%, 60%, and 42%, respectively. Furthermore, paxilline was shown to decrease heart rate in a dose-dependent manner. These results implicate BK channels located in the heart to be directly involved in the regulation of heart rate.

Structural determinants of lolitrems for inhibition of BK large conductance Ca2+-activated K+ channels

Lolitrem B is an indole-diterpenoid neurotoxin which is the main causative agent of ryegrass staggers, an animal disease associated with tremors and incoordination. It is also a potent inhibitor of large conductance calcium-activated potassium (BK) channel activity (IC(50)=4 nM). Furthermore, we have recently shown that the motor function deficits induced by lolitrem B are specifically mediated by BK channels, making the toxin a valuable tool for investigating the molecular function and physiological roles of these channels. To determine what structural features of BK channel agents are required for high potency, the effect of lolitrem B and seven structurally-related lolitrems on BK channel activity has been measured. Concentration-responses and conductance-voltage (G-V) relationships were determined for each compound and related to the different structure types. This study has identified seven new BK channel inhibitors and has allowed the identification of two key structural features required for high potency BK channel activity by lolitrems.

The evaluation of endophyte toxin residues in sheep fat

Abstract 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 evaluation of fungal endophyte toxin residues in milk

Abstract AIM: To determine the concentrations of fungal endophyte toxins in the milk of cows fed perennial ryegrass containing wild-type or AR37 endophyte. METHODS: Groups of 10 multiparous Holstein-Friesian cows were fed wild-type (containing lolitrem B) or AR37 (containing epoxy-janthitrems) endophyte-infected perennial ryegrass (Lolium perenneL.). Animals were kept indoors and fed for 12 days. Over this period, animals were regularly assessed for ryegrass staggers and herbage intake measured. At the conclusion of the 12-day indoor-feeding period, cows were grazed on AR1 (toxin-free) pastures for a further 8 days. Daily individual milk samples and milk yields were collected over the complete 20-day period. Milk samples were analysed for endophyte toxins using HPLC methods developed during this study. Daily herbage samples were also taken and concentrations of endophyte toxins measured. RESULTS: Methods were successfully developed for the analysis of lolitrem B and epoxy-janthitrems in milk which allowed the concentrations of these compounds in milk to be compared with the concentrations in feed consumed by the animals. Both toxin types could be detected in milk after only 1 day of exposure to respective treatment pastures. The maximum concentration of endophyte toxins in milk was 5 ng/mL lolitrem B and 109 ng/mL epoxy-janthitrems from cows fed wild-type and AR37 endophyte-infected ryegrass pastures, respectively. Concentrations of epoxy-janthitrems present in herbage were much higher than for lolitrem B (Day 1–12 average of 14.6 and 1.8 ppm, respectively). Despite the high concentrations of epoxy-janthitrems consumed by cows fed AR37 endophyte-infected pastures no signs of ryegrass staggers were observed over the experimental period, whereas those cows fed wild-type endophyte-infected pastures all showed signs of ryegrass staggers. This is consistent with the view that epoxy-janthitrems are low potency tremorgens. At the conclusion of the toxin feeding period, endophyte toxin concentrations in milk quickly dropped to almost zero after 8 days. A comparison of the quantities of lolitrem B and epoxy-janthitrems consumed by each cow with the quantities secreted in milk showed that only very low proportions of the total amount ingested are secreted in milk (0.23% lolitrem B and 0.49% epoxy-janthitrems). CONCLUSION: Lolitrem B and epoxy-janthitrems can be detected in the milk of cows consuming wild-type and AR37 endophyte-infected perennial ryegrass, respectively. Concentrations detected were low and changed quickly in association with the amounts being consumed by the cows. Available evidence gives no indication that these compounds may pose a threat to human health.

Deletion and gene expression analyses define the paxilline biosynthetic gene cluster in Penicillium paxilli.

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

Mechanism of action of lolitrem B, a fungal endophyte derived toxin that inhibits BK large conductance Ca2+-activated K+ channels

The aim of this study was to compare the mode of action of the commonly used BK inhibitor paxilline with that of the more recently discovered lolitrem B. Similarities and differences in characteristics of inhibition between the two compounds were investigated. We have previously shown that lolitrem B does not affect the BK channel G-V, in contrast to the rightward shift produced by paxilline. These different effects on the voltage-dependence of activation suggest different modes of action for these two compounds. In this study we show that inhibition by both paxilline and lolitrem B is characterized by an open state preference for BK (hSlo) channels. Both compounds had a 3-fold higher apparent affinity under conditions likely to favour the open state, suggesting they have a similar BK conformational preference for binding. Furthermore, both compounds had a calcium concentration-dependence to their inhibitory effects. The G-V shift induced by paxilline was calcium concentration-dependent.

Temperature and Plant Genotype Alter Alkaloid Concentrations in Ryegrass Infected with an Epichloë Endophyte and This Affects an Insect Herbivore.

Asexual Epichloë endophytes colonize agricultural forage grasses in a relationship which is mutually beneficial and provides the host plant with protection against herbivorous insects. The endophyte strain AR37 (Epichloë festucae var. lolii) produces epoxy-janthitrem alkaloids and is the only endophyte known to provide ryegrass with resistance against porina larvae (Wiseana cervinata (Walker)), a major pasture pest in cooler areas of New Zealand. This study examined the effect of temperature on concentrations of epoxy-janthitrems in AR37-infected ryegrass and determined how the resulting variations in concentration affected consumption, growth and survival of porina larvae. Twenty replicate pairs of perennial (Lolium perenne L.) and Italian ryegrass (L. multiflorum Lam.) plants with and without endophyte were prepared by cloning, with one of each pair grown at either high (20°C) or low (7°C) temperature. After 10 weeks, herbage on each plant was harvested, divided into leaf and pseudostem, then freeze dried and ground. Leaf and pseudostem material was then incorporated separately into semi-synthetic diets which were fed to porina larvae in a bioassay over 3 weeks. Epoxy-janthitrem concentrations within the plant materials and the semi-synthetic diets were analyzed by high performance liquid chromatography. AR37-infected ryegrass grown at high temperature contained high in planta concentrations of epoxy-janthitrem (30.6 μg/g in leaves and 83.9 μg/g in pseudostems) that had a strong anti-feedant effect on porina larvae when incorporated into their diets, reducing their survival by 25–42% on pseudostems. In comparison, in planta epoxy-janthitrem concentrations in AR37-infected ryegrass grown at low temperature were very low (0.67 μg/g in leaves and 7.4 μg/g in pseudostems) resulting in a small anti-feedant effect in perennial but not in Italian ryegrass. Although alkaloid concentrations were greatly reduced by low temperature this reduction did not occur until after 4 weeks of exposure. Alkaloid concentrations were slightly lower in Italian than in perennial ryegrass and concentrations were higher in the pseudostems when compared with the leaves. In conclusion, epoxy-janthitrems expressed by the AR37 endophyte show strong activity against porina larvae. However, when ryegrass plants are grown at a constant low temperature for an extended period of time in planta epoxy-janthitrem concentrations are greatly reduced and are less effective against this pasture pest.