Epichloë fungal endophytes – a vital component for perennial ryegrass survival in New Zealand

ABSTRACT

Pastoral agriculture underpins the New Zealand economy. Its success in generating export income is due to a mild and moist temperate climate, productive soils, innovative farmers supported by an effective research and development system, and an energy efficient production system based on year-round grazing. However, pastoral agriculture is entirely reliant on introduced pasture and forage species and their ability to withstand both endemic and introduced pasture pests. Critical to this is the mutualistic relationship between ryegrass and Epichloë fungal endophytes which provide a range of secondary metabolites that deter herbivory by both ruminants and insect pests. The challenge has been to identify and commercialise Epichloë strains which while providing protection against insect pests, ensuring ryegrass persistence, do not cause animal health and welfare issues. The critical role that Epichloë fungal endophytes play in maintaining pasture persistence and as a result the pastoral economy of New Zealand will be reviewed.

KEYWORDS:

• Alkaloids
• drought
• endophytes
• grass
• insect pests
• persistence

Introduction

New Zealand’s economy is heavily reliant on pastoral agriculture to provide food and fibre products that constitute up to 50% of exports by value (OECD 2019; Ministry of Primary Industries 2021) and between 5 and 6% of GDP (Statista 2022). This value generation is a consequence of a mild and moist temperate climate, productive soils, and grassland pastures capable of year-round grazing. These, along with innovative farmers, have ensured that New Zealand’s pastoral agricultural systems are among the most efficient in terms of energy use and greenhouse gas emissions (Ledgard et al. 2007; Clune et al. 2017; Leahy et al. 2019; Ledgard et al. 2021; Beef+LambNZ 2022; Mazzetto et al. 2022) and this is despite the long shipping distances involved for supply to international markets (Mazzetto et al. 2023). However, an often overlooked and understated factor which ensures industries that are reliant on pastoral production in New Zealand are viable is the association grassland plants have with some mutualistic fungal endophytes (Caradus and Johnson 2020; Caradus et al. 2021a). This review will provide an overview of the pastoral agricultural system in New Zealand, the role of grasses, their strengths and weaknesses, and the critical part that Epichloë fungal endophytes play in maintaining pasture persistence, and how more beneficial Epichloë strains have been developed and commercially delivered.

The New Zealand pastoral ecosystem

The indigenous vegetation ecosystems of New Zealand were a mosaic of rainforest, swampland, and tussock, but with European arrival in the mid-nineteenth century the transformation of landscapes began leading to the development of extensive grassland ecosystems consisting of introduced species (Lancashire 2006; Caradus et al. 2021a). While there was some milling of native forest for timber, much of the clearing came from extensive, and at times, indiscriminate burning (Wolfe 2022). Similarly, considerable amounts of swamp land were drained (McGlone 2009).

Prior to European arrival, endemic grassland in New Zealand made up about 82 432 km² or 31% of the total land area, with the majority found in the South Island (57%) (Mark and McLennan 2005). There were five types of grassland identified with 13% being low-alpine snow tussock grassland, 18% montane to subalpine snow tussock grassland, 23% montane to low-alpine tall red/copper tussock grassland, 44% montane to subalpine short-tussock grassland, and 2% lowland sward grassland. In the early 2000s, about 44% of indigenous grasslands persisted, albeit in a variously modified or degraded condition (Mark and McLennan 2005). However, between 2001 and 2008 the rate of conversion of endemic grassland into other uses was 4600 ha per year which is greater than between 1990 and 2001 when it was 3,500 ha per year (Weeks et al. 2013). Only 12.3% is currently assigned by the Department of Conservation as formally protected (Mark and McLennan 2005).

New Zealand’s pastoral systems are now dominated primarily by European grass and forage species, but in subtropical northern regions also include kikuyu (Pennisetum clandestinum) from highland east Africa and paspalum (Paspalum spp.) from South America (Percival 1977, 1978). Predominant European grass species include perennial ryegrass (Lolium perenne), annual ryegrass (L. multiflorum), tall fescue (Fescue arundinacea) and cocksfoot (Dactylis glomerata); legumes include white clover (Trifolium repens), red clover (T. pratense) and lucerne (Medicago sativa); and herbs include chicory (Cichorium intybus) and plantain (Plantago lanceolata). Perennial ryegrass germplasm was first brought from the United Kingdom in the early 1800s (Stewart 2006). It was not until the 1930s that modern breeding of perennial ryegrass began in earnest. This was based on using ecotypes that had developed within New Zealand, selecting elite genotypes and crossing these with germplasm from mild oceanic regions of Northwest Spain to provide improved winter growth and late flowering. Crossing with Italian ryegrass to produce ‘short rotation hybrids’ with improved winter growth, and then backcrossing to perennial ryegrass to develop ‘long rotation hybrids’ also has occurred. Tetraploid perennial and annual ryegrasses have also been used extensively, most derived from diploid New Zealand cultivars (Stewart 2006).

The value of perennial ryegrass

Using direct and industry dependent Gross Domestic Product contributions, perennial ryegrass has been estimated to be the most valuable plant species in New Zealand, from an economic impact viewpoint. It is valued at 3.3 times the value of the second most valuable species Pinus radiata, and over six times the value of white clover, the third most valuable species (Nixon 2016).

Perennial ryegrass is the most widely adapted perennial forage grass in temperate regions due to its high digestibility and tolerance of grazing (Wilkins 1991). While it is adapted to moist, cool temperate conditions it is generally considered to be moderately to poorly tolerant of prolonged periods of low soil moisture availability (Levy 1970; Tozer et al. 2011). However, ryegrass as a species is considered well adapted to the New Zealand climate (Hunt and Easton 1989; Easton et al. 2001; Chapman et al. 2011), with a high digestibility and nutritive value (Powell et al. 1978; Wilman and Riley 1993), and as a plant it is well adapted for year-round grazed pastoral farming which dominates the New Zealand landscape.

Threats to perennial ryegrass persistence

Persistence, considered by farmers as the most important factor limiting pasture performance (Smith and Brazendale 2011), can result from the loss of both sown plants and overall yield (Parsons et al. 2011; Dodd et al. 2018). Perennial ryegrass, despite being the dominant grass in New Zealand pastures, has several abiotic and biotic threats to both its yield and persistence. These include drought caused by low rainfall and higher temperatures, a range of insect pests, and a recently legislated nitrogen fertiliser cap of 190 Kg N per ha (Mfe 2022; New Zealand Legislation 2022). Nitrogen fertilisation has been shown to increase leaf number, leaf dry weight/plant and tiller number/plant leading to improved persistence in intensively grazed pasture (Harris et al. 1996). Soil fertility needs to be reasonably high for perennial ryegrass to reach acceptable yields (Lambert et al. 1986). Drought is a major driver for perennial ryegrass decline caused by interactions between levels of summer rainfall, the water holding capacity and texture of the soil, along with plant nitrogen status (Clark 2011). Changes in climate resulting in more pronounced drought and higher temperatures are likely to be detrimental to ryegrass in the future (Lee et al. 2013; Kalaugher et al. 2017; Beukes et al. 2021; McCahon et al. 2021).

Pathogens such as crown and stem rusts (Puccinia coronata and P. graminis) are the predominant leaf pathogens found on perennial ryegrass in New Zealand (Corkill 1956; Skipp and Hampton 1996). Barley yellow dwarf virus can also be widespread in perennial ryegrass swards more than 2 years old (Latch 1977, 1980). Insect pests (Goldson et al. 2005; Ferguson et al. 2019), particularly in combination with drought and over-grazing, have a significant impact on perennial ryegrass persistence (Clark 2011; Zydenbos et al. 2011; Hewitt et al. 2021) (Table 1).

Table 1. Insect pests of perennial ryegrass in New Zealand and their impact.

Insect pest	Damage	Estimated magnitude of impact ($m/year)	References
Grass grub (Costyletra giveni)	Root-feeding larvae cause visible damage at densities >200/m2; up to 50% loss of ryegrass	Dairy – $140 to $380 Sheep and beef – $75 to $205	Zydenbos et al. 2011; Ferguson et al. 2019
African black beetle (Heteronychus arator)	Adults destroy seedling ryegrass; Second and third instar larvae feed on grass roots close to the crown; >20/m2 are damaging	Dairy – $148 to $223 Sheep and beef – $15 to $19	Bell et al. 2011; Ferguson et al. 2019
Porina (Wiseana species)	Noticeable damage with densities > 40/m^2; larval damage caused by grazing leaf tissue	Dairy – $70 to $84 Sheep and beef – $78 to$ 88	Ferguson et al. 2019
Argentine stem weevil (ASW) (Listronotus bonariensis)	Adult ASW can destroy ryegrass seedlings; damage to mature ryegrass relatively insignificant but increases with N application. Most damage caused by larval mining of tillers.; up to 20% loss of ryegrass	Dairy – $100 to $130 Sheep and beef – $60 to $70	Hunt 8. Gaynor 1982; Zydenbos et al. 2011; Ferguson et al. 2019
Root aphid (Aploneura lentisci)	Can reduce ryegrass yields due to feeding on phloem in roots	Not documented, but becoming considered as being significant	Popay and Gerard 2007; Popay and Cox 2016; Muller 2019; Popay et al. 2021
Pasture mealy bug (Balanococcus poae and other species)	Reduce yields and impacts plant survival	Not documented, but can impact ryegrass survival particularly in the South Island	Pennell and Ball 1999; Pennell et al. 2005
Black field cricket (Telegryllus commodus and Lepidogryllus sp.)	Populations of 20 crickets/m^2 could cause pasture loss of 1600 kg DM/ha, primarily during February–April; damage caused by 60 crickets/ha equivalent to 21 ewes per ha	Not documented, but at high population densities will be significant	Blank and Olsen 1979, 1981, 1987; Blank et al. 1985; Blank 1986
Various root invading nematodes	Comprised of several species in 3 genera, Pratylenchus, Heterodera and Meloidogyne. Significant impact on legume component of pastures; but does affect grass yields	Dairy – $274 Sheep and beef – $326	Watson et al. 1986; Yeates and Barker 1986; Ferguson et al. 2019

Epichloë the vital ingredient for perennial ryegrass persistence

The mutualistic obligate endophyte Epichloë has been identified as an essential component of perennial ryegrass ensuring its persistence against many abiotic and biotic challenges (Johnson et al. 2013; Caradus and Johnson 2020; Hewitt et al. 2021). Asexual Epichloë endophytes have no documented free-living stage in nature and are vertically transmitted through host seeds (Philipson 1989). However, intervention has allowed Epichloë isolates to be cultured and then re-infected into new and possibly quite different temperate grass germplasm (Latch and Christensen 1985). It is unlikely that two strains of Epichloë will exist together within the same grass genotype (Christensen et al. 2000). The survival of Epichloë endophytes in the seed can be reliant on storage conditions with viability reducing if stored at high temperatures and high humidity (Tian et al. 2013), and even if stored at ambient temperatures will decline significantly after only 6 months (Hume et al. 2011). Storage of seed containing Epichloë is recommended to be at low temperatures (less than 5°C) and low relative humidity (less than 60%) (Rolston et al. 1986).

The success of Epichloë in protecting perennial ryegrass is largely due to the secondary metabolites (including alkaloids) that the endophyte produces within the plant. The known secondary metabolites fall into four groups (Bush et al. 1997; Schardl et al. 2012, 2013; Saikkonen et al. 2013; Panaccione et al. 2014):

1. Ergot alkaloids – which can be divided into four groups based on their chemical structure: clavines (e.g. chanoclavine, agroclavine), lysergic acid, lysergic acid amides (e.g. ergonovine, ergine), and ergopeptines (e.g. ergovaline, ergotamine, ergocornine, ergocristine, ergosine, ergocryptine);

2. Indole diterpenoids – lolitrems, epoxyjanthitrems, terpendoles, paxilline;

3. Lolines – N-formyl loline (NFL), N-acetyl loline (NAL), N-acetylnorloline (NANL); and

4. Pyrrolopyrazine – peramine.

These can have a range of effects on both grazing animals and/or insect pests (Table 2). Notably, ergot alkaloids and indole-diterpenoids affect both ruminants and some insects while pyrrolopyrazines, and loline alkaloids have little or no effect on grazing ruminants but do deter or kill insect pests. The variation between Epichloë strains for alkaloid production provides an opportunity to select strains that reduce animal health and welfare issues and yet protect the plant against insect pests. Popay and Hume (2011) summarised the effect of different endophyte strains on a range of insect pests and concluded that ‘Argentine stem weevil is controlled by all endophytes [commercialised at that time] except perhaps Bealey NEA2; black beetle is controlled by all endophytes except AR1; pasture mealybug is controlled by AR1, AR37 and Standard and likely to also be controlled by Endo5 and NEA2; porina and root aphid are controlled by AR37’. For some insect pests, such as African black beetle, there are few insecticidal options and control is largely due to Epichloë endophyte and good pasture management (Ferguson et al. 2019).

Table 2. Effects of secondary metabolites produced in perennial ryegrass by Epichloë endophytes and their effects on ruminants and biotic challenges to perennial ryegrass persistence and yield.

Secondary metabolite	Effect on ruminants	Reference	Effect on biotic challenges	References
Ergot alkaloids
Ergocryptine	Vasoconstriction causing heat stress	Klotz et al. 2010	Antifeeding effect on African Black Beetle adults	Ball et al. 1997a
			Antifeeding effect on both adult and larva ASW	Dymock et al. 1989
Ergotamine	Vasoconstriction causing heat stress	Klotz et al. 2007	Antifeeding effect on African Black Beetle adults	Ball et al. 1997b
			Antifeeding effect on adult ASW	Dymock et al. 1989
Ergovaline	Vasoconstriction causing heat stress	Fletcher 1993a; Fletcher 1993b; Easton et al. 1996; Bluett et al. 2001; Caradus et al. 2022	Antifeeding effect on African Black Beetle adults, and reduce oviposition	Prestidge et al. 1994; Ball et al. 1997a; Popay and Ball 1998
	Reduced feed intake by ruminants	Edwards et al. 1993; Pennell and Rolston 2003; Caradus et al. 2022 (refers to pers. com. from RH Watson)	Antifeeding effect on ASW adults but not larvae	Popay et al. 1990; Popay and Wyatt 1995; Popay and Ball 1998
	Depressed live weight gain	Fletcher et al. 1991; Fletcher and Sutherland 1993; Panaccione et al. 2006
Indole-diterpenoid alkaloids
Epoxyjanthitrems	Intense but short lived staggers	Fletcher and Sutherland 2009; Babu et al. 2018	Reduction of survival of root aphids by AR37 – effect of epoxyjanthitrem is unproven	Popay et al. 2004; Popay and Gerard 2007; Popay et al. 2021
			Reduces feeding and survival of porina	Jensen and Popay 2004; Popay et al. 2012
Lolitrem B	Severe staggers; increased respiration rate, heart rate and blood pressure	Miles et al. 1992; Munday-Finch et al. 1995, 1996; Guerre 2016	Feeding deterrent and toxin of ASW larvae, but not adults	Popay et al. 2003a
			Possibly reduces root-knot nematode infection	Ball et al. 1997a
Paxilline	Moderate tremorgen	McLeay and Smith 1999	Feeding deterrent for ASW larvae	Rowan 1993
			Reduces feeding and weight gain of porina	Babu 2008
			Attractant of slugs (Deroceras spp.)	Barker 2008
Terpendole C or M	Mild tremorgen	Munday-Finch et al. 1997; Gatenby et al. 1999	No recorded activity against insect pests
Pyrrolopyrazine alkaloid
Peramine	No known mammalian toxicity	Pownall et al. 1995	Feeding deterrent for both ASW adults and larvae; reduced oviposition	Rowan et al. 1990
Loline alkaloids
N-acetyl loline (NAL)	No known mammalian toxicity	Schardl et al. 2007; Finch et al. 2017	Reduced root aphid numbers per plant	Schmidt and Guy 1997; Jensen and Popay 2007
			Strong grass grub larvae deterrent	Popay and Tapper 2007
N-acetylnorloline (NANL)	No consistent mammalian toxicity	Schardl et al. 2007; Finch et al. 2017	Increased mortality of ASW larvae	Jensen et al. 2009; Popay et al. 2009
			Weak deterrent of grass grub larvae	Popay and Tapper 2007
N-formyl loline (NFL)	No known mammalian toxicity	Schardl et al. 2007; Finch et al. 2017	Reduced root aphid numbers per plant	Schmidt and Guy 1997; Jensen and Popay 2007
			Argentine stem weevil larvae: increased mortality	Jensen et al. 2009; Popay et al. 2009
			Grass grub larvae: strong deterrent	Popay and Lane 2000; Popay and Tapper 2007
			Lesion nematode: repellent and toxic	Kimmons et al. 1990; Bacetty et al. 2009
Lolines collectively – not defined	No known mammalian toxicity	Schardl et al. 2007; Finch et al. 2017	Reduce grass grub larvae feeding and weight gain; deterrent effect	Popay and Lane 2000; Popay et al. 2003b; Patchett et al. 2008b, 2011
			Feeding deterrent of black field cricket	Barker et al. 2015a;
			African black beetle: antifeeding effect on larval and adult stages	Bryant et al. 2010; Barker et al. 2015a
			Argentine stem weevil: feeding deterrent and death of larvae; reduces oviposition	Patchett et al. 2008a; Jensen et al. 2009; Barker et al. 2015b
			Porina larvae: reduced feeding and weight gain	Popay and Lane 2000
			Root aphid: reduced populations	Schmidt 1993
			Deroceras slug: reduced feeding	Barker 2008

While largely a research curiosity, documented evidence of Epichloë antifungal activity against economically important fungal pathogens is extensive (Card et al. 2021). However, these effects have rarely been exploited commercially and no correlation has been demonstrated between antifungal bioactivity of endophyte strains and their known alkaloid profiles (Siegel and Latch 1991; Christensen 1996). Three possible mechanisms to consider when contemplating the effect of Epichloë endophytes on fungal pathogens include: (a) antibiosis caused by non-alkaloid secondary metabolites (summarised in Card et al. 2021), (b) induced resistance by a beneficial microbe such as Epichloë resulting in reduced disease susceptibility of the plant, and (c) competition for limiting factors such as physical space or metabolites (Zabalgogeazcoa 2008; De Kesel et al. 2021). No accounts of Epichloë effects on bacterial phytopathogens have been documented (Card et al. 2021).

Effects of Epichloë endophyte secondary metabolites on improving the tolerance of perennial ryegrass to abiotic stresses are less well documented than those for biotic stresses. However, studies have shown that associations of Epichloë with temperate grasses can in some circumstance improve the host plant’s tolerance/ resistance to drought (Barker et al. 1993; Hahn et al. 2008; Hewitt et al. 2021), salinity (Reza Sabzalian and Mirlohi 2010; Yin et al. 2014), nutrient poor soils (Malinowski and Belesky 1999; Malinowski et al. 2000), and heavy metals (Mirzahossini et al. 2015).

Naturally occurring Epichloë endophytes in perennial ryegrass do not express loline secondary metabolites (Ball and Tapper 1999; Schardl et al. 2007). The expression of lolines in perennial ryegrass by Epichloë can only be achieved by inoculating endophyte strains from other temperate grasses, and in particular fescues, into ryegrass. However, the survival and transmission of Epichloë endophyte is reliant on genetic compatibility between host plant and endophyte strain (Christensen et al. 1997). This becomes a challenge when strains are moved across taxa (Schardl et al. 2004; Caradus and Johnson 2020) and while achievable the levels of transmission and viability in seed have not allowed successful commercialisation at scale (Caradus and Johnson 2020). Additionally, the expression levels of loline in ryegrass are often low and variable (Easton et al. 2006).

Delivering Epichloë strains as commercial products

In some regions of New Zealand, Epichloë endophyte is essential for perennial ryegrass persistence (Johnson et al. 2013; Johnson and Caradus 2019; Caradus and Johnson 2020; Hewitt et al. 2021). However, because some endophyte strains contain alkaloids that cause animal health concerns (Table 2), the challenge for seed companies seeking to commercialise effective Epichloë strains is to identify strains expressing secondary metabolites that do not cause animal health and welfare issues but do provide protection against insect pests. Several Epichloë endophyte strains have been commercialized in New Zealand for use with perennial ryegrass (Table 3).

Table 3. Description of commercialised Epichloë strains for perennial ryegrass (adapted from Caradus and Johnson 2020; Caradus et al. 2021a and b). Note that the protection provided against insect pests is not necessarily due to the known alkaloids expressed.

Brand name of strain	Known alkaloids	Protection provided
Standard	High ergovaline, peramine and lolitrem B	Argentine stem weevil, pasture mealybug, African black beetle; root aphid
AR1	High peramine	Argentine stem weevil and pasture mealy bug
AR37	High epoxyjanthitrems	Argentine stem weevil (adult), pasture mealybug, porina, African black beetle, root aphid
Endo5	Medium ergovaline, high peramine	Argentine stem weevil, pasture mealy bug, African black beetle, root aphid
NEA	Low ergovaline and peramine, very low lolitrem B	Pasture mealybug, African black beetle
NEA2	Medium ergovaline, medium-low peramine, very low lolitrem B	Argentine stem weevil, pasture mealybug, African black beetle
NEA4	Medium ergovaline, medium-low peramine, very low lolitrem B	Argentine stem weevil, pasture mealybug, African black beetle
Edge	Medium peramine	Argentine stem weevil, pasture mealy bug
Happe	Loline compounds – NFL, NAL, and NANL	African black beetle adult, root aphid, pasture mealybug, porina
U2	Loline compounds – NFL, NAL, and NANL	African black beetle, Argentine stem weevil, pasture mealybug, root aphid, grass grub, field crickets

The uptake of these novel endophyte strains in New Zealand has been high (Milne 2007; Caradus et al. 2013), driven by the need to solve a significant on farm issue of reducing animal health and welfare issues caused by the Standard wild type endophyte strain (Easton et al. 1996; Fletcher et al. 1999), while protecting the grass plant against insect pests (Popay and Hume 2011; Zydenbos et al. 2011). The ease of use and application also ensured farmer acceptance and use of this technology. The economic impact in New Zealand of AR37 alone, which provides tolerance to a wide range of insect pests (Table 3), has been estimated at NZ$3.6 billion over 20 years (ACIL Allen Consulting 2017). AR37 provides significant benefits to sheep farmers through providing improved growth during the summer and autumn with lambs averaging over a 6-year period, 44 g/head/day on Standard endophyte, 129 g/day on nil-endophyte and 131 g/day on AR37 infected pastures, representing increases in lamb growth of 198% over Standard endophyte (Fletcher 2005). At the on-farm level use of AR1 has also been shown to increase animal liveweight gains, resulting in 22% returns over farms using Standard endophyte pastures (Fletcher 1999), and a 9% (Bluett et al. 2003) and 14% (Ussher 2003) increase in milk solids was measured in dairy systems.

Well-designed production and quality assurance guidelines are an integral part of delivering a high-quality endophytic seed technology, and in doing so giving the farmer confidence that it will provide the promised benefits (Rolston and Agee 2007). Endophyte viability in seed should be above 70% at the point of sale to ensure farmers are purchasing a quality product (Hume and Barker 2005; Easton and Tapper 2008). This can be achieved by using appropriate packaging and seed storage conditions (Rolston et al. 1986). In New Zealand, the majority of proprietary ryegrass seed is sold with one of the novel endophytes, only small amounts of nil endophyte and Standard endophyte seed is sold (Caradus et al. 2013, 2021b).

Concluding comment

The success of perennial ryegrass in driving the agricultural economy of New Zealand has been significantly influenced by its association with the obligate fungal mutualist Epichloë, the consequence of which was only begun to be understood from the early 1980s. As the biology of Epichloë became better documented the opportunity to identify more beneficial strains has resulted in the successful commercialisation of strains with reduced animal health and welfare impacts and improved protection against insect pests. Improved environmental outcomes are also possible if future gene editing of endophyte is permitted under regulation to allow targeted manipulation of alkaloid pathways and insertion of genes capable of expressing compounds that reduce methane emissions. The ultimate outcome is for Epichloë to be a natural biocontrol to protect grasses from pest and disease challenges and improve yield in drought conditions without needing to resort to the use of synthetic chemistry.

Disclosure statement

No potential conflict of interest was reported by the author(s).