Alison J. Popay

A symbiosis expressed non‐ribosomal peptide synthetase from a mutualistic fungal endophyte of perennial ryegrass confers protection to the symbiotum from insect herbivory

While much is known about the biosynthesis of secondary metabolites by filamentous fungi their biological role is often less clear. The assumption is these pathways have adaptive value to the organism but often the evidence to support this role is lacking. We provide the first genetic evidence that the fungal produced secondary metabolite, peramine, protects a host plant from insect herbivory. Peramine is a potent insect feeding deterrent synthesized by Epichloë/Neotyphodium mutualistic endophytes in association with their grass hosts. The structure of peramine, a pyrrolopyrazine, suggests that it is the product of a reaction catalysed by a two‐module non‐ribosomal peptide synthetase (NRPS). Candidate sequences for a peramine synthetase were amplified by reverse transcription polymerase chain reaction. Four unique NRPS products were identified, two of which were preferentially expressed in planta. One of these hybridized to known peramine producing strains. This clone was used to isolate an Epichloë festucae cosmid that contained a two‐module NRPS, designated perA. Nine additional genes, which show striking conservation of microsynteny with Fusarium graminearum and other fungal genomes, were identified on the perA‐containing cosmid. Associations between perennial ryegrass and an E. festucae mutant deleted for perA lack detectable levels of peramine. A wild‐type copy of perA complemented the deletion mutant, confirming that perA is a NRPS required for peramine biosynthesis. In a choice bioassay, plant material containing the perA mutant was as susceptible to Argentine stem weevil (ASW) (Listronotus bonariensis) feeding damage as endophyte‐free plants confirming that peramine is the E. festucae metabolite responsible for ASW feeding deterrent activity.

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.

Occurrence and impact of pasture mealybug (Balanococcus poae) and root aphid (Aploneura lentisci) on ryegrass (Lolium spp.) with and without infection by Neotyphodium fungal endophytes

Abstract Pasture mealybug (Balanococcuspoae) was found infesting two field trials evaluating the performance of selected strains of the endophyte Neotyphodium lolii in ryegrass (Lolium spp.) in Canterbury, New Zealand. Deterioration of endo‐phyte‐free plots relative to endophyte‐infected plots had been observed. In Trial A, pasture mealybug were sampled in plots of the perennial ryegrass cultivar ‘Grasslands Nui’, without endophyte (nil), or infected with the wild‐type endophyte, or the selected strains, AR1 and AR37. In Trial B, mealybug numbers on six ryegrass cultivars infected with AR1 or wild‐type were compared with those on the same cultivars without endophyte. In sampling these trials, the presence of a root aphid, Aploneura lentisci, was also noted. Populations of mealybug in both trials were similar on all endophyte‐infected treatments and significantly lower than populations on nil treatments. Neither AR1 nor wild‐type appeared to reduce root aphid numbers, while AR37 may have had some effect. The amount of dead grass was significantly greater in nil than in endophyte‐infected plots in Trial A, and yield of ryegrass was correlated with numbers of mealybug and root aphid present. Pasture productivity in nil treatments had recovered by spring, in the year of the outbreak. The presence of endophyte, strain of endophyte and cultivar were all significant factors affecting both total dry matter and green yield in Trial B. Pasture mealybug accounted for 55% of the variation in a decline in growth rate that occurred in this trial over the summer‐autumn periods between 2000 and 2001, particularly in the nil treatments. Two years after the outbreak there was 25% less ryegrass and persistently lower pasture yields in nil plots. We conclude that pasture mealybug are capable of inflicting severe damage to endophyte‐free ryegrass in Canterbury, particularly during dry summer‐autumn periods.

New Zealand pest management: current and future challenges.

New Zealand is under increasing pressure from terrestrial and aquatic pests, weeds and diseases that threaten the countrys ecosystems and economy. Ongoing improvement in existing pest management methodologies and novel approaches are required in response to public concerns about animal welfare, increasingly stringent trade requirements, abolition of groups of pesticides and resistance to existing pesticides as well as, possibly, biological control agents. Surveillance and pest monitoring are needed to increase the chances of early interception of invasive species or to confirm their eradication. Core capabilities in taxonomy, genomics, phenology, ecology, pest impacts, development of novel control tools and social science are required and must be maintained nationally. Given New Zealands unique environment, the ecology of invasive pests cannot be presumed to be the same as that in their native ranges, yet currently many pests in New Zealand are managed with poor understanding of their bionomics and impacts. Failure to address these areas will have serious adverse impacts on New Zealand.

Evaluating the performance of endophytes in farm systems to improve farmer outcomes – a review

Abstract. The main plant species relied on for forage supply to grazing animals in New Zealand and south-eastern Australia is perennial ryegrass (Lolium perenne L.). Perennial ryegrass has evolved with a fungal endophyte (Neotyphodium lolii, Latch, Christensen & Samuals) that occupies intercellular spaces, and is nourished by its host. The endophyte (referred to as standard or wild-type) provides the plant with protection from a range of insect pests by producing alkaloids, some of which are also toxic to grazing animals, causing ryegrass staggers and/or exacerbating heat stress. Over the last 20 years naturally occurring perennial ryegrass endophytes have been found in Europe that produce less of the alkaloids that cause animal health problems but have similar or enhanced effects as the standard endophyte on deterring insect attack on infected plants, when introduced into New Zealand and Australian-bred ryegrasses. This review provides a summary of endophyte research in New Zealand from the perspective of insect pests, plants (particularly perennial ryegrass) and the animals grazed on ryegrass-dominant pastures. The protocols used to evaluate perennial ryegrass/endophyte associations over the past 30 years are also discussed. Future testing of new grass/endophyte associations should include the utilisation of more environments for agronomic and entomological experiments; routinely carrying out small animal toxicology assays, and the running of short-term indoor feeding experiments with sheep and cows. Implementation of these changes provides the minimum requirements for strengthening the evaluation of new endophyte associations so farmers using these technologies, gain optimal benefits from their adoption.

Plant-endophyte-herbivore interactions: More than just alkaloids?

A recent paper by Rasmussen et al. (New Phytol 2007; 173:787-97) describes the interactions between Lolium perenne cultivars with contrasting carbohydrate content and the symbiotic fungal endophyte Neotyphodium lolii at different levels of nitrogen supply. In a subsequent study undertaken by Rasmussen et al. (Plant Physiol 2008; 146:1440-53) 66 metabolic variables were analysed in the same material, revealing widespread effects of endophyte infection, N supply, and cultivar carbohydrate content on both primary and secondary metabolites. Here, we link insect numerical responses to these metabolic responses using multiple regression analysis. Addendum to: Rasmussen S, Parsons AJ, Bassett S, Christensen MJ, Hume DE, Johnson LJ, Johnson RD, Simpson WR, Stacke C, Voisey CR, Xue H, Newman JA. High nitrogen supply and carbohydrate content reduce fungal endophyte and alkaloid concentration in Lolium perenne. New Phytol 2007; 173:787-97. and Rasmussen S, Parsons AJ, Fraser K, Xue H, Newman JA. Metabolic profiles of Lolium perenne are differentially affected by nitrogen supply, carbohydrate content, and fungal endophyte infection. Plant Physiol 2008; 146:1440-53.

Effect of different Neotyphodium endophytes on root distribution of a perennial ryegrass (Lolium perenne L.) cultivar

Abstract Plants of perennial ryegrass either endophyte‐free or infected with Neotyphodium lolii endophyte strain AR1, AR37 or wild‐type, were grown in 105‐mm‐diameter tubes of sand in a glasshouse. Root angles were determined by counting root intercepts on semi‐circular stainless steel mesh transects running from near the edge of the tube at the sand surface to directly under the plant. The average number of root intercepts did not vary between endophyte strains or between their average and the endophyte‐free treatment. However, the linear decline of root intercept frequency with depth varied according to the endophyte status of the ryegrass. Endophyte‐infected plants had fewer roots close to the surface. This effect of shoot endophytes on root distribution patterns in ryegrass may improve the plants tolerance of moisture stress.

Field resistance to Argentine stem weevil (Listronotus bonariensis) in different ryegrass cultivars infected with an endophyte deficient in lolitrem B

Abstract A spaced plant trial, a small plot trial, and a field trial evaluated an endophyte (Acremonium spp.) (AR6) which does not produce lolitrem B in the tetraploid cultivars Grasslands Moata (Lolium multiflorum) and Grasslands Greenstone (L. multiflorum × perenne), and in the perennial cultivars (L. perenne) Grasslands Pacific and Grasslands Nui. The endophyte was compared to three lolitrem‐producing isolates viz. AR41, Waiau, and the wildtype. In all trials Argentine stem weevil (ASW) (Listronotus bonariensis) adult feeding, oviposition, and larval damage were recorded at regular intervals between October and March. ASW attack was highest on the tetraploid cultivars, Greenstone and Moata. In most instances, all endophytes reduced adult ASW feeding and oviposition in the different cultivars compared with their respective endophyte‐free (E‐) controls. Feeding and oviposition on endophyte‐infected (E+) Moata and Greenstone was in some instances similar to, or higher than, that on E‐ Nui or Pacific. All ...

Endophyte Isolate and Host Grass Effects on Chaetocnema pulicaria (Coleoptera: Chrysomelidae) Feeding

ABSTRACT Endophytic fungi belonging to the genus Neotyphodium, confer resistance to infected host grasses against insect pests. The effect of host species, and endophtye species and strain, on feeding and survival of the corn flea beetle, Chaetocnema pulicaria Melsheimer (Coleoptera: Chrysomelidae) was investigated. The grass—endophyte associations included natural and artificially derived associations producing varying arrays of common endophyte-related alkaloids or alkaloid groups, peramine, lolitrem B, ergovaline, and the lolines. Preference and nonpreference tests showed that C. pulicaria feeding and survival were reduced by infection of tall fescue with the wild-type strain of N. coenophialum, the likely mechanism being antixenosis rather than antibiosis. In the preference tests, endophyte and host species effects were observed. Of the 10 different Neotyphodium strains tested in artificially derived tall fescue associations, eight strongly deterred feeding by C. pulicaria, whereas the remaining two strains had little or no effect on feeding. Infection of tall fescue with another fungal symbiont, p-endophyte, had no effect. Perennial ryegrass, Lolium perenne L., infected with six strains of endophyte, was moderately resistant to C. pulicaria compared with endophyte-free grass, but four additional strains were relatively inactive. Six Neotyphodium—mendow fescue, Festuca pratensis Huds., associations, including the wild-type N. uncinatum—mendow fescue combination, were resistant, whereas three associations were not effective. Loline alkaloids seemed to play a role in antixenosis to C. pulicaria. Effects not attributable to the lolines or any other of the alkaloids examined also were observed. This phenomenon also has been reported in tests with other insects, and indicates the presence of additional insect-active factors.

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.