Cool season grasses of the family Poaceae harbour fungal endophytes of the genus Epichloë. Asexual Epichloë endophytes grow as unbranched hyphae within the above ground tissues of the host plant and are transmitted between reproductive generations within the seed of its host. There is an ongoing debate over the nature of the relationship between endophytes and their host (Saikkonen et al., 1998, 2010). The relationship between agricultural forage grasses and asexual Epichloë endophytes, however, is thought to be defensive mutualistic. Defensive mutualism was first proposed by Clay (1988) and involves both organisms benefiting from the relationship. The endophyte gains from its host shelter, nutrients and a means of transmission (Saikkonen et al., 2004). In return the plant gains increased protection from biotic stresses including insects (Prestidge et al., 1982; Ball and Prestidge, 1992; Pennell et al., 2005; Popay et al., 2012), mammalian herbivores (Edwards et al., 1993; Cosgrove et al., 2002), pathogens (Pańka et al., 2013) and nematodes (Eerens et al., 1997; Bacetty et al., 2009) as well as increased tolerance to abiotic stresses such as drought and nutrient stress (Ravel et al., 1997; Kane, 2011; Nagabhyru et al., 2013).

Plants infected with an asexual Epichloë endophyte can have increased resistance against herbivorous insects due to the production of alkaloids which can have anti-feedant and/or toxic effects (Rowan et al., 1990; Jensen et al., 2009; Popay et al., 2009). Understanding bioactive alkaloids, their distribution within the plant and their effects on insects enables endophytes to be used in pest management strategies in both farming systems and turf. Fungal endophytes have been recognized as an important part of New Zealand’s pastoral sector since the early 1980s, as New Zealand contains a number of herbivorous pasture pests which can cause severe pasture damage.

The common toxic endophyte (Epichloë festucae var. lolii) strain found naturally infecting ryegrass (Lolium perenne and L. boucheanum syn. L. hybridum) in New Zealand produces alkaloids which provide the host with protection against a number of important pest insects (Prestidge et al., 1982; Popay and Baltus, 2001; Pennell et al., 2005). It also, however, produces lolitrem B an alkaloid which causes ryegrass staggers, a neurological impairment (Cunningham and Hartley, 1959; Fletcher and Harvey, 1981; di Menna et al., 2012) and the alkaloid ergovaline which causes vasoconstriction in grazing livestock (Dyer, 1993; Klotz et al., 2007). Due to these harmful effects on livestock endophyte research in New Zealand has focused on identifying different E. festucae var. lolii strains from European grasslands, where there is a greater chemical diversity, in an attempt to select those with a favorable chemical profile. Endophyte strains that are found to produce beneficial alkaloids, to deter insects, but not the detrimental alkaloids are then inoculated into New Zealand ryegrass cultivars (Johnson et al., 2013). These strains are known as ‘selected endophytes.’ One selected strain of E. festucae var. lolii is AR37. The only known alkaloids to be produced by AR37 are the epoxy-janthitrems (Tapper and Lane, 2004; Finch et al., 2007, 2012), a group of five compounds within the indole diterpene class of alkaloids. The epoxy-janthitrems are lipophilic compounds and are not easily translocated around the plant. Therefore, concentrations are thought to be highest in the pseudostem where endophyte mycelia are concentrated. AR37 provides ryegrass with protection against many of New Zealand’s major ryegrass pests including; African black beetle adults [Heteronychus arator (F.) Coleoptera: Scarabaeidae] (Ball et al., 1994), Argentine stem weevil larvae [Listronotus bonariensis (Kuschel), Coleoptera: Curculionidae] (Popay and Wyatt, 1995), root aphid [Aploneura lentisci (Passerini), Aphididae: Fordinae] (Popay et al., 2004; Popay and Gerard, 2007) and porina larvae (Wiseana spp. Hepialidae: Lepidoptera) (Jensen and Popay, 2004).

Porina are a group of seven closely related moth species endemic to New Zealand. The larvae of many of these species are a pest of cultivated grasses (Dugdale, 1994), particularly in the lower half of the North Island and in many parts of the South Island of New Zealand. Temperature is one of the main environmental factors which influences the location of porina in New Zealand. A study by Allan et al. (2002) looked at survival of larvae to pupation and then adulthood at four temperatures. Larval survival was found to be significantly lower when larvae were grown at 20°C compared to those grown at both 10 and 15°C. But survival was higher at 20°C than 5°C. Porina larvae are nocturnal and emerge at night from vertical burrows created beneath the soil surface (Barlow et al., 1986). Larvae can be highly destructive as they feed by cutting ryegrass tillers off at the base of the plant or by grabbing low lying leaves before dragging the herbage back into their burrow (Harris, 1969). The ‘selected’ endophyte AR37 has been shown to provide ryegrass with resistance against porina larvae in pot trials (Jensen and Popay, 2004), choice bioassays (Jensen and Popay, 2004) and field trials (Popay et al., 2012). In addition, when pure and semi-pure epoxy-janthitrem I, produced by AR37, was incorporated into a semi-synthetic diet and fed to porina larvae, larval diet consumption and growth were significantly reduced (Finch et al., 2010; Hennessy, 2015).

Several abiotic and biotic factors including plant growth temperature (Ball et al., 1995; Eerens et al., 1998; Salminen et al., 2005) and plant genotype (Adcock et al., 1997; Easton et al., 2002; Faeth et al., 2002) are known to affect alkaloid concentrations within endophyte-infected ryegrass. What effect these factors have on epoxy-janthitrem concentrations in ryegrass is not known. In this paper, the results of two experiments, a ryegrass pot trial and a porina larval bioassay, were designed to investigate the effect of high (20°C) and low (7°C) growth temperature on epoxy-janthitrem concentrations in AR37-infected perennial (L. perenne L.) and Italian (L. multiflorum Lam.) ryegrass and to examine how any resulting variations in concentration would affect consumption, growth and survival of porina larvae.

This experiment has shown that when AR37-infected ryegrass was grown at 20°C epoxy-janthitrem concentrations were greatly increased, resulting in a strong anti-feedant effect on porina larvae that led to high weight loss and in the case of pseudostems, increased mortality. In contrast, epoxy-janthitrem concentrations declined markedly in plants grown at 7°C causing low level deterrence and a small reduction in weight gain of larvae fed perennial ryegrass. Although epoxy-janthitrem concentrations were greatly reduced by low temperature this reduction did not occur until after 4 weeks of exposure.

When fed to larvae E+ perennial ryegrass grown at LT reduced larval consumption and growth but Italian ryegrass did not. This is likely explained by the higher epoxy-janthitrem concentrations in perennial ryegrass insect diets, although this effect was exaggerated by the large increase in consumption and growth of larvae fed E- perennial ryegrass (cv ‘Grasslands Samson’) that did not occur in larvae fed E- Italian ryegrass (cv ‘Grasslands Asset’). It is possible that differences in the ratios of the five epoxy-janthitrem compounds between perennial and Italian ryegrass may have contributed to the differences in bioactivity, particularly if certain compounds, or combinations of compounds are more bioactive than others. It is also possible that there was an unknown alkaloid produced in higher concentrations in perennial than Italian ryegrass.

Results from this study have shown an anti-feedant effect of the endophyte AR37 on porina larvae when ground herbage was incorporated into an insect diet. Epoxy-janthitrems within AR37 are likely to be responsible for this bioactivity as pure and semi-pure epoxy-janthitrem I have previously been shown to have an anti-feedant effect on porina when incorporated into semi-synthetic diets (Finch et al., 2010; Hennessy, 2015).

Although the results from this experiment clearly show an anti-feedant effect of AR37 it could not be determined whether this endophyte also has a toxic effect on larvae. Here toxicity is defined as a reduction in growth and survival of an insect above that which can be attributed to starvation. Pseudostems of AR37-infected ryegrass grown at HT, which contained the highest epoxy-janthitrem concentrations, reduced larval survival. A reduction in survival could indicate toxicity but it is also possible that larvae may have died due to starvation caused by the strong anti-feedant effect of AR37. Further research is required to resolve this.

Plant growth temperature is known to affect the concentrations of other important endophyte alkaloids. Seasonal concentrations of lolitrem B, which like the epoxy-janthitrems is in the indole diterpene class of alkaloids, and peramine were monitored by Ball et al. (1991). Lolitrem B concentrations were found to be highest during the summer months and lowest during the winter when rainfall is higher and temperatures are cooler. Peramine concentrations were comparatively stable, but were also significantly lower during winter when compared to summer and autumn. Although caution must be applied when relating results of pot trials to field conditions the results of this study suggest that epoxy-janthitrems could respond to temperature in a similar way. However, for epoxy-janthitrem concentrations to decrease to the low levels observed in this experiment plants would have to be exposed to constant low temperatures for an extended period of time (at least 4 weeks). Under field conditions temperatures will constantly fluctuate which may mean that epoxy-janthitrem concentrations are not decreased to the extent as that observed in this study.

The reduction in epoxy-janthitrem concentrations in plant material grown at low temperatures suggests that AR37 may not provide the highest level of protection against porina larvae during the winter months in parts of New Zealand. Porina are major pasture pests particularly in the southern areas of both the North and South Island of New Zealand where they are capable of causing severe pasture damage. Several species of porina are known pasture pests, the moths of which have different peak flight periods. Moths of W. cervinata, the species tested in this experiment, fly between October and December in the South Island (Barratt et al., 1990). Young larvae of this species will begin feeding on ryegrass during the late spring and summer months, when temperatures are warm. Results from this study suggest that during this period AR37-infected ryegrass is likely to contain relatively high epoxy-janthitrem concentrations which should provide good control over larvae. Larvae of the later flying species, W. copularis, which can fly as late as February (Barratt et al., 1990) begin feeding on AR37-infected ryegrass when temperature and alkaloid concentrations are likely to be lower and less effective at controlling larval populations.

The mechanisms by which temperature and plant genotype affected alkaloid concentrations in perennial and Italian ryegrass plants in this study are not known. These factors may have indirectly affected alkaloid concentrations by influencing the ratio of endophyte mycelial biomass to plant biomass, resulting in changes in alkaloid concentration (di Menna and Waller, 1986; Breen, 1992; di Menna et al., 1992; Ju et al., 2006). Alternatively, alkaloid biosynthesis, metabolism, or degradation rates may have been directly affected by temperature or plant genotype (Spiering et al., 2005).

No published information is available comparing epoxy-janthitrem concentrations in the leaves and pseudostems of AR37-infected ryegrass plants. In this study, concentrations were found to be markedly higher in the pseudostems than the leaves at both temperatures and for both cultivars. This distribution is not uncommon and has also been found for lolitrem B (di Menna et al., 1992; Davies et al., 1993; Keogh et al., 1996; Ball et al., 1997). Alkaloids such as lolitrem B and the epoxy-janthitrems are lipophilic compounds and are not easily translocated around the plant (Ball et al., 1993; Munday-Finch and Garthwaite, 1999; Spiering et al., 2005) thus distribution tends to be similar to that of the endophyte, which is generally higher in the pseudostem and lower in the leaves (Musgrave, 1984; Musgrave and Fletcher, 1984; Keogh and Tapper, 1993). Maintaining high alkaloid concentrations in the pseudostem is advantageous for both the host plant and the endophyte as the meristem, the tissue containing undifferentiated cells and where growth occurs is located at the base of the ryegrass plant (Popay, 2009). Tiller death will occur if an insect severely damages the meristem. Insect damage to the leaves of ryegrass plants is not as harmful to the plant itself, as ryegrass is adapted to animal grazing (Popay, 2009). However, the more leaf material the insect is able to consume the less that is available for both plant photosynthesis and consumption by grazing livestock, resulting in reduced plant growth and animal productivity.

The endophyte AR37 is very important for the control of porina in New Zealand as although other endophytes such as AR1 and the common toxic strain provide protection against some pest insects (Prestidge et al., 1982; Popay et al., 1999; Pennell et al., 2005; Popay and Gerard, 2007; Popay and Thom, 2009) it is only AR37 which provides ryegrass with protection against porina (Jensen and Popay, 2004; Popay et al., 2012). Control against porina, which are a major pasture pest in parts of New Zealand, currently involves an integrated pest management strategy involving planting ryegrass infected with the AR37 endophyte and the application of insecticides at particular times of the year (Barratt et al., 1990). The results of this paper support the continued use of integrated pest management strategies to control porina populations in the field.

Leading on from this study field trials should be conducted to determine how temperature affects epoxy-janthitrem concentrations in AR37-infected ryegrass in the field and how these concentrations then impact on porina populations. If concentrations are found to be reduced under certain environmental conditions the next step could be to identify existing ryegrass cultivars and/or plant genotypes, from which a new breeding line could be produced, that produces higher alkaloid concentrations when grown at low temperature.

LH carried out this research as a part of her Masters of Science (Research). AP was a co-supervisor and the main supervisor of all experimental work. SF was a co-supervisor and oversaw all of the chemical analyses. MC was the University supervisor and VC provided statistical expertise.

AP is a patent holder for AR37; AP and SF receive research funding from IP owners Grasslanz Technology Ltd. and licensee, PGG Wrightson Seeds. AP receives a share of royalties from the sale of AR endophytes. All the other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.