APPLICATION FORM RELEASE

Application to import for release or to release from containment new organisms under the Hazardous Substances and New Organisms Act 1996

Send by post to: Environmental Protection Authority, Private Bag 63002, Wellington 6140 OR email to: [email protected]

Application number

APP201774

Applicant

Grasslanz Technology Ltd and AgResearch Ltd

Key contact

John Caradus

www.epa.govt.nz 2

Application to import for release or to release from containment new organisms

Important

This application form is to seek approval to import for release or release from containment new organisms (including genetically modified organisms). The application form is also to be used when applying to import for release or release from containment new organisms that are or are contained within a human or veterinary medicine. Applications may undergo rapid assessment at the Authority’s discretion if they fulfil specific criteria. This application will be publicly notified unless the Authority undertakes a rapid assessment of the application. This application form will be made publicly available so any confidential information must be collated in a separate labelled appendix. The fee for this application can be found on our website at www.epa.govt.nz. If you need help to complete this form, please look at our website (www.epa.govt.nz) or email us at [email protected]. This form was approved on 1 May 2012.

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Application to import for release or to release from containment new organisms

1. Brief application description Provide a short description (approximately 30 words) of what you are applying to do. To release non-toxic Neotyphodium fungi in order to improve the resistance of rye corn and other annual cereal crops to pests and diseases, reducing pesticide and fungicide use and improving farm productivity.

2. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it.

New Zealand produces approximately 1 million tonnes of grains for industry and another 1.3 million tonnes for silage from cereal crops every year. Grown on 150,000 hectares the total value of sales of grain and silage from New Zealand’s arable farmers is estimated at $645 million which contributes $713 million to the country’s GDP (Sanderson et al. 2012). These crops are used by industry for milling (flour, malting and stock feed) and by dairy and other livestock farmers as silage. New Zealand forage grasses are often infected with epichloae fungi that live within the plant (endophytes), and which produce compounds that protect pastures from insect pests. Without these fungi, pastoral agriculture would not be possible in large parts of New Zealand due to damage from insect pests. Endophyte technology has been a recent focus of AgResearch and we have now developed the most comprehensive endophyte capability world- wide. Animal-safe endophytes have now been successfully commercialised and today contribute approximately $200 million per annum to the New Zealand economy (Johnson et al. 2013). Endophyte technology is not available for cereals which are increasingly being used as forage in New Zealand. This means that many modern cereals lack epichloae endophytes that could improve their resistance to insect pests and fungal diseases. AgResearch, as a leader in this area, has recently managed to form symbiotic associations in containment between Neotyphodium fungal endophytes and rye corn (Secale cereale). Initial testing has shown promise and these associations are now ready to move from containment. It is important to New Zealand that our farmers remain internationally competitive. Furthermore, there is ongoing pressure from consumers, both domestic and international, to reduce the amount of synthetic chemicals, pesticides and fungicides used to produce food. We believe that New Zealand would benefit from the introduction of endophytes that can help improve the resistance of cereal crops to pests and diseases. Our aim is to reduce synthetic chemical use, which benefits the environment as well as reducing cost to farmers. In additional, these fungi have been known to improve the drought tolerance of infected plants. It is our assessment that the release of these Neotyphodium species will provide significant and meaningful benefits to farmers in New Zealand. The Neotyphodium species we are applying to import can only be introduced into host plants under laboratory conditions. They are also asexual and therefore incapable of spreading between plants other than via seeds. The fungi colonise seed from host plants, thereby spreading ‘vertically’. The species in this application do not produce toxins that affect mammals and pose no risk to humans or livestock. We have identified no risks in the release of these fungi. Closely related species are already in New Zealand. For example, N. lolii, N. coenophialum, N. uncinatum, and N. occultans endophytes are common in New Zealand pastures (Easton 2001; Johnson et al. 2013). These endophytes are now considered essential components of sustainable grass based pasture ecosystems (Hill et al. 2005). Furthermore, these species are widely recognised as safe, with risks further reduced by their inability to spread between plants, even including that of the same species.

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Application to import for release or to release from containment new organisms

3. Describe the background and aims of the application This section is intended to put the new organism(s) in perspective of how they will be used. You may use more technical language but please make sure that any technical words used are included in a glossary.

Epichloë and Neotyphodium fungi, collectively known as epichloae, are natural associates of many grass genera and tribes of the subfamily Pooideae (Schardl et al. 2004), including ryegrasses that are important to New Zealand (Easton 2007; Easton and Fletcher 2007). The specific characteristics of associations vary (Faeth and Saikkonen 2007; Rudgers and Clay 2007), but the fungus commonly enhances the fitness of the host by increasing tolerance of water and nutrient limitations (Malinowski et al. 2005; Malinowski and Belesky 2000), and by protecting them from a range of invertebrate pests and diseases (Popay and Bonos 2005). This is mediated by bioactive compounds, and in terms of the effect on invertebrate pests, some are very well characterised (Bush et al. 1997; Lane et al. 2000), and others are recently described (Tapper and Lane 2004). Since 1980, research on grass- endophyte associations, and development of new associations, have transformed pasture agronomy and livestock husbandry (Easton and Fletcher 2007; Johnson et al. 2013). Many cool-season grasses (Poaceae, subfamily. Pooideae) which possess seed-borne Neotyphodium fungal endophytes are known for their bioprotective properties, and especially for production of anti-pest alkaloids such as lolines (Zhang et al. 2010) and peramine (Koulman et al. 2007). In particular loline alkaloids and the alkaloid peramine confer such protection without notable or known toxicity to mammals or humans consuming the grass or products derived indirectly from consumption of the grass (see section 6 below). The primary aim is to provide cereal crops with the ability to deter insect pests and disease via the production of fungal secondary metabolites and reduce the use of synthetic pesticides and fungicides. Strains, many of which produce the metabolites peramine and lolines that are not toxic to mammals will be employed in this work. We seek to use these non-stroma forming epichloae fungal endophytes in modern cereals outside of containment. Stroma are required for spread of the fungus by production of sexual spores, but these do not occur in Neotyphodium endophytes. Their taxonomy is still being developed as on-going discoveries are made of these endophytes in wild relatives of cultivated pasture grasses and cereals. Neotyphodium endophytes are asexual, they do not produce sexual spores, meaning they cannot spread from one plant to another by means of infectious ascospores or undergo genetic change via sexual recombination (Schardl, 2010). The associations formed by Neotyphodium endophytes are defined as mutualistic as these endophytes can enhance their hosts’ survival through protection from abiotic and biotic stresses. Our aim is to utilise the mutually beneficial epichloae/grass symbioses in an agricultural context for modern cereals. Some pasture grass epichloae species have been present in New Zealand for over 150 years. With these species we have been successful in developing and commercialising animal-safe grass endophyte associations that confer bio-protective properties for increased pasture (ryegrass and fescue) persistence and productivity. We wish to extend this to crops such as rye corn (Secale) and other annual cereal crops. To-date we have successfully created, under containment conditions, symbioses between annual cereal crops and endophytes isolated from wild Triticeae grasses collected from Asia, Eurasia, Middle East, Eastern Europe, North America and South America. AgResearch has a longstanding research programme examining the fungal endophytes of pasture grasses. These fungal symbionts accompanied their grass hosts when perennial ryegrass was first imported into New Zealand around 200 years ago (Stewart 2006). The strains that persist from those original imports can be toxic to both mammalian and invertebrate consumers of the grass (Easton 2007). The ecotype Neotyphodium lolii in New Zealand produces the neurotoxin lolitrem B and the vasoconstrictive toxin ergovaline (Bluett et al. 2005). Through our research we have sourced and identified strains of this class of fungus (Neotyphodium spp.) that do not produce key mammalian toxins but retain the ability to produce compounds that are toxic and/or repellent to invertebrate pasture pests (Easton et al. 2001).

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Application to import for release or to release from containment new organisms

Modern cereals are not naturally infected with epichloae fungi, however, they are found in their wild relatives. Leveraging off our pasture grass expertise with endophytes we have infected cereal grasses, such as wheat, barley, rye corn, Triticale and oats, with non-toxic endophytes with a view to providing non-synthetic chemical pest protection to cereal field crops. These endophytes will enhance cereal resistance to pests and diseases, providing a more reliable and cost-effective feed for livestock industries. In New Zealand, pesticide use on cereals for grain, silage or baleage alone is estimated to be about 1.6 T of active ingredient per year and for fungicides about 12 T of active ingredient per year (Chapman 2010). However, the use of many pesticides can be problematic due to the known problems associated with the chemicals frequently used for such purposes. Many pesticides are toxic and can be dangerous to human and animal consumers of treated agricultural crops (Casida and Quistad 1998). In particular, the accumulation in humans and animals of toxic pesticides can lead to serious health issues for individuals, particularly during early development. For example, pesticide exposure has been linked to respiratory disorders, developmental cancers, and has been shown to have lasting effects on mental development (Zejda et al. 1993). The use of pesticides may be difficult to control in variable environmental conditions leading to unwanted dispersal of toxic compounds, for example by drift of sprays or by soil leaching. In addition, pests may develop pesticide resistance for a number of reasons, including improper practice and handling, and this poses a real threat to crop (grain) yields. Accordingly, there is a need for pest control measures that do not use applied pesticides. Therefore, the primary aim of this release application is to provide cereal crops with the ability to deter pests and diseases via the production of fungal secondary metabolites. Fungal endophyte strains that produce the metabolites peramine and lolines, which are documented as non-toxic to mammals, will be employed in this work (Ball and Barker et al. 1997; Schardl 2010). Lolines are known to be generally pest-deterring compounds produced in grasses infected by some epichloae strains. Schardl et al. (2007) concluded that “in vivo studies employing aphids have consistently supported a role for lolines both in deterrence and insecticidal activity.” Another study (Dahlman et al. 1997) gave additional examples of insecticidal activity of N-formyl loline not mentioned by Schardl et al. (2007). Lolines have been shown to increase resistance of the host grass plants to pest herbivory (Bush et al. 1997). The specific lolines may have some variations in the bioactivities against specific pests. It has also been suggested that the presence of lolines may provide a host plant with some level of protection from environmental stresses including drought and spatial competition (Malinowski and Belesky 2000). Peramine (a pyrrolopyrazine alkaloid) is a bioactive alkaloid produced by some combinations of endophytes and grasses (Schardl et al. 2012). Peramine production has been shown to be dependent upon the functioning of at least one gene of endophyte origin (Tanaka et al. 2005). Peramine has been shown to be a feeding deterrent of some insects which cause damage to grasses (Rowan and Latch 1994). Our aim is to improve production systems involving cereal grasses by reducing the need to apply synthetic chemical control agents for plant pest and disease control by using a naturally occurring endophytic symbiont that has a proven track record in grass based pasture systems (Easton et al. 2001; Kuldau and Bacon 2008). An object of this application is to provide Neotyphodium fungal endophyte strains which, when combined with annual cereal crops such as Secale, confer economically significant (i.e. the benefits are non-negligible) pest protection to the Secale plant.

4. Information about the new organism(s)

 Provide a taxonomic description of the new organism(s) (if the organism is a genetically modified organism, provide a taxonomic description of the host organism(s) and details of the genetic modification).  Describe the biology and main features of the organism including if it has inseparable organisms.  Describe if the organism has affinities (e.g. close taxonomic relationships) with other organisms in New Zealand.

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Application to import for release or to release from containment new organisms

 Could the organism form an undesirable self-sustaining population? If not, why not?  What is the ease with which the organism could be eradicated if it established an undesirable self-sustaining population?

Taxonomic description of the new organism Non-GMOs: Neotyphodium species (Order Hypocreales, Family Clavicipitaceae) The fungal endophytes in this application have been isolated from wild relatives of modern cereals (Elymus and Hordeum). The strains for which we are seeking release from containment are listed in Table 1. These strains result in the production of the compounds loline, peramine, chanoclavine and terpendole E in the host plant. These are not all the strains that have been discovered within the Neotyphodium group, but are the strains that have the appropriate biological activity based on known chemistry to make an impact on pest and disease resistance of the cereal host plant, while not conferring additional risks to mammals or the environment. The table contains the reference number assigned to each endophyte strain identified; the accession number used in the Margot Forde Germplasm Centre for the plant material from which the endophyte strain was identified; the species name of the germplasm from which the endophyte was identified; the geographic origin of the host germplasm; and the known alkaloids expressed by the endophyte strain when in the host plant germplasm.

Table 1. Description of Neotyphodium strains that are the subject of this application

Alkaloids present and expressed by the Germplasm endophyte in planta Endophyte accession number Host species from which the Geographic origin

strain of population from endophyte strain was of the host reference which the

identified and isolated germplasm number endophyte was sourced Peramine Chanoclavine E Terpendole Lolines AR3002 BZ2155 Elymus dahuricus China AR3005 BZ2159 Elymus sp. China AR3007 BZ2162 Elymus dahuricus China Hordeum brevisubulatum spp. AR3013 BZ10534 violaceum Iran AR3014 BZ10535 Hordeum bogdanii Kazakhstan AR3015 BZ2162 Elymus dahuricus China AR3017 BZ2162 Elymus dahuricus China AR3019 BZ2155 Elymus dahuricus China AR3020 BZ2160 Elymus sp. China AR3023 BZ2162 Elymus dahuricus China AR3029 BZ4455 Hordeum bogdanii China AR3035 BZ4455 Hordeum bogdanii China AR3039 BZ2679 Elymus caninus UK AR3042 BZ2162 Elymus dahuricus China AR3045 BZ2162 Elymus dahuricus China AR3046 BZ4833 Elymus mutabilis Kazakhstan AR3048 BZ4833 Elymus mutabilis Kazakhstan AR3049 BZ4833 Elymus mutabilis Kazakhstan

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AR3050 BZ4833 Elymus mutabilis Kazakhstan AR3051 BZ4820 Elymus virginicus USA AR3052 BZ4820 Elymus virginicus USA AR3053 BZ4820 Elymus virginicus USA AR3054 BZ4820 Elymus virginicus USA AR3055 BZ4820 Elymus virginicus USA AR3059 BZ4815 Elymus canadensis USA AR3061 BZ4969 H. bogdanii China AR3064 BZ4952 Elymus mutabilis Russia AR3065 BZ4897 Elymus ciliaris China AR3068 BZ5339 Elymus mutabilis Russia AR3070 BZ5473 Elymus dahuricus subsp. excelsus Mongolia AR3071 BZ5474 Elymus dahuricus subsp. excelsus China AR3073 BZ5510 Elymus caninus unknown AR3074 BZ5564 Elymus caninus Russia AR3075 BZ5578 Elymus elymoides ssp. brevifolius Canada AR3076 BZ5589 Elymus mutabilis var. oschensis Estonia AR3078 BZ5592 Elymus nevskii Russia AR3079 BZ5602 Hordeum bogdanii China AR3080 BZ5085 Elymus varius China AR3081 BZ5628 Elymus nevskii unknown AR3082 BZ5473 Elymus dahuricus subsp. excelsus Mongolia AR3083 BZ5473 Elymus dahuricus subsp. excelsus Mongolia AR3084 BZ5510 Elymus caninus unknown AR3087 BZ5598 Elymus scabrifolius Argentina AR3088 BZ5598 Elymus scabrifolius Argentina AR3089 BZ5076 Elymus pendulinus Russia

Biology and main features of the organism Epichloae (Epichloë/Neotyphodium species) endophytes are fungal symbionts of cool-season grasses that form long term systemic infections that vary within the symbiotic continuum from antagonism to mutualism and can be transmitted vertically (via seed) and horizontally (from plant to plant) via ascospores (Bush et al. 1997). Epichloae are ascomycetes (family Clavicipitaceae) with a bipolar heterothallic mating system, forming fungal stroma, an external mycelial structure that gives rise to spermatia. A fly of the genus Phorbia, not present in New Zealand, is responsible for the transfer of these spermatia (Schardl et al. 2012).

Epichloae endophytes have three distinct dispersal mechanism types; type I where stroma are obligatory on infected plants (Epichloë), type II where stroma are optional (Epichloë), and type III (Neotyphodium) where no stroma is formed (Leuchtmann and Clay 1997). Neotyphodium endophytes are asexual derivatives of Epichloë and similarly infect a number of cool season grasses of the order Pooideae (Clay 1993; Schardl 1996; Christensen et al. 2002). The asexual Neotyphodium form asymptomatic mutualistic symbioses with their hosts, and transmit vertically via host seed colonisation (Schardl and Clay 1997). In the vertical transmission route of asexual and pleiotropic epichloae endophytes the fungus invades the developing ovule and ultimately the embryo of mature seeds (Philipson and Christey 1986). In this clonal and highly efficient means of propagation of the fungus, nearly 100% of seeds from infected mother plants transmit the endophyte (Siegel et al. 1984).

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The associations that the various Neotyphodium species form are host specific. Neotyphodium lolii specifically colonises perennial ryegrass (Lolium perenne), N. coenophialum colonises tall fescue (L. arundinaceum syn. Schedonorus phoenix syn. Festuca arundinacea), N. uncinatum colonises meadow fescue (Festuca pratensis) and N. occultans colonises annual grasses such as L. multiflorum.

It is thought that speciation of the host has progressed alongside that of the symbiont fungus, contributing to a co- speciation that manifests in this host specificity (Schardl et al. 1997). It is suggested that multiple infections from sexual Epichloë spp. have given rise to hybrid asexual endophyte species (Schardl et al. 1991) that essentially became trapped in their host species. Using molecular techniques, Schardl et al. (1991) showed that multiple copies of TUB2 genes are present in many Neotyphodium, suggesting that the different species have developed by super-infection and hybridisation while within their host grasses (Schardl and Clay 1997).

Neotyphodium endophytes are obligate symbionts. They have no known capacity to exist independently of their host grasses in nature. However, it is possible to isolate them from surface disinfected plant tissue in the laboratory and culture them on simple agar media such as potato dextrose agar (PDA) (Latch and Christensen 1985). The symbioses that Neotyphodium form are mutualistic in that both the fungus and the host grass benefit from the association. The fungus benefits from a biological niche with few if any competing organisms and a ready source of nutrients in the host apoplastic fluid along with a mechanism for vicarious dispersal via the host seed. The host benefits from the range of secondary metabolites the fungus produces in the form of alkaloids, many of which have individual and/or multiple activities against different classes of organisms.

As with the Epichloë, four classes of specific alkaloids produced only in the host plant have received intensive study in grasses hosting Neotyphodium endophytes. These are the pyrrolizidines (lolines), ergot alkaloids (clavines (including chanoclavine), lysergic acids and derivative alkaloids), indole diterpenoids (lolitrems and terpendoles including terpendole E) and pyrrollopyrazine (peramine) alkaloids (Siegel and Bush 1997). Lolines, clavines and peramine alkaloids individually or in combination can offer endophyte-infected grasses considerable advantage over endophyte-free grasses in that they can confer insect pest resistance and protection to the host. In Neotyphodium lolii/Lolium perenne associations in New Zealand insect pest resistance is the primary advantage achieved via the production of peramine and ergovaline. Peramine is associated with resistance to the pasture pest Argentine stem weevil (Listronotus bonariensis) (Prestidge et al. 1991) while ergovaline is associated with resistance to African black beetle (Heteronychus arator) (Ball and Miles et al. 1997). The tall fescue endophyte N. coenophialum confers primary advantage via drought resistance and is capable of extending the southern range limit of tall fescue grasses in agricultural areas of the southern part of North America. The research into this phenomenon has examined both direct physiological effects of the endophyte symbiont on the physiology of the host plant affecting stomatal conductance and osmotic adjustment (Elm and West 1995) and indirect effects via differences in nematode populations affecting the host plant (West et al. 1987).

Affinities (e.g. close taxonomic relationships) with other organisms in New Zealand Epichloae feature prominently in New Zealand agriculture in imported grasses such as perennial ryegrass, tall fescue, annual/Italian ryegrass (L. multiflorum), and meadow fescue. This class of fungus also has affinities at the genus level with an endemic endophyte in Poa matthewsii (Stewart et al. 2004) and a native endophyte in Echinopogon ovatus (Moon et al. 2002). Fungal endophytes of this type have been associated with endemic and native flora for some time. There is no evidence at all of the ability to hybridise; indeed Schardl (Schardl 2010) places Neotyphodium aotearoae (ex Echinopogon ovatus) in its own clade. There has been no similar characterisation of the endophytes in Poa matthewsii. Neotyphodium endophytes are important in agricultural grazing systems that involve cool season grasses such as the above mentioned perennial ryegrass in New Zealand and tall fescue in the United States (Woodfield and Easton 2004). In the New Zealand context, Neotyphodium endophytes have been shown to confer advantages to their host plants via the production of secondary metabolites that have activity against invertebrates (Prestidge and Gallagher 1988) and, contrary to the aims of agriculture, grazing mammals (Smith and Towers 2002). In addition,

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Application to import for release or to release from containment new organisms there is evidence that endophyte infection of host grasses can confer resistance to nematodes (Bacetty et al. 2009) and drought resistance (Elm and West 1995). Ryegrass staggers is caused by the neurotoxin lolitrem B, one of several alkaloid metabolites produced by endophyte-infected perennial ryegrass of the New Zealand ecotype (Fletcher and Harvey 1981). Ergovaline is also produced in endophyte-infected New Zealand ecotype ryegrass and can result in low live weight gains, reduced milk production, and general ill-thrift of grazing animals (Fletcher 1999, Butendieck et al 1994). A solution to this problem has been to source Neotyphodium endophytes with a range of alkaloid phenotypes (chemotypes) including those with anti-insect alkaloids but either no or low impact anti- mammalian alkaloids. Plant hosts containing such endophytes have been obtained from Europe and collections in the USA. These strains can be isolated in the laboratory and inoculated into novel hosts (Latch and Christensen 1985; Easton 2007).

Likelihood of forming an undesirable self-sustaining population Rye corn is a domesticated cereal crop. It is grown under conditions that provide tilled and fertile soils along with intensive management of weeds, and pests and fertilisers. Rye corn is grown as an annual crop, at the end of the season no plants are left remaining in the field. For a self-sustaining population to establish it would have to develop outside of the confines of the cropping field where establishment is unlikely given the competitive environments with other plants, pests, diseases and the absence of any active cultivation or management. The establishment of novel endophyte-infected populations requires laboratory-based isolation and culture of the fungus from infected plants and inoculation into new plant hosts using a specialised technique. This is because the fungus colonises above ground plant tissues including the ovaries through which it colonises the embryo and is vertically transmitted through the seed. Self-sustaining populations will not form spontaneously in other potential hosts. In nature these fungi do not survive independently of their host grasses (i.e. they have no free living stage: Figure 1) and cannot form self-sustaining populations. Figure 1. The life cycle of Neotyphodium endophytes (http://www.grasslanz.com/UnderstandingtheScience/novelendophytetechnologies.aspx)

Ease of eradication Rye corn and other cereal crops are readily eradicated with trans-located herbicides such as glyphosate or contact herbicides. All of these crops are annuals where seed is harvested as grain.

The organism that is the subject of this application is also the subject of: a. an innovative medicine application as defined in section 23A of the Medicines Act 1981. Yes X No b. an innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997. Yes X No

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5. Detail of Māori engagement (if any)

Discuss any engagement or consultation with Māori undertaken and summarise the outcomes. Grasslanz Technology believes that the endophytes, for which we are seeking release, will be of significant benefit to Māori cereal cropping farmers through improved drought tolerance and reduced inputs of pesticides and fungicides. Incorporation of endophyte-infected cereals within Māori farming systems would be conducive to Māori ethical and cultural ideals of sustainability, low chemical input, and resilience. In May 2013 Grasslanz Technology Ltd wrote to over 300 Māori groups listed on the Maori National Network. By early November 2013, four responses had been received and these are summarised below. The full submissions are appended. Submission from Paul Elwell-Sutton, from Snapshot Creek, Haast, Westland raised 3 issues: 1. Evidence that Neotyphodium endophytes cannot change genetically 2. Evidence that Neotyphodium species cannot spread horizontally 3. Clarify whether the endophyte strains to be released have been created using any genetic engineering technology. Our response to Paul Elwell-Sutton – 1. Neotyphodium endophytes are the asexual morphs of the broader Epichloë genus. As such they have no sexual phase which could provide the opportunity for genetic change (Schardl et al. 1997). As with many organisms, spontaneous mutations can occur resulting in genetic change – but this is also a natural process, and frequency of change can be extremely slow. 2. Closely related species of these Neotyphodium endophytes (i.e. other members of the Neotyphodium genus) have been part of New Zealand’s ryegrass pastures for more than 100 years. During that time there has been no documented horizontal spread of Neotyphodium endophytes within or between species. They complete their whole life cycle within the plant as shown in Figure 1. 3. None of the strains in this application have been developed using any genetic engineering techniques. They are all natural strains of Neotyphodium species isolated from natural populations of wild grasses.

Submission from Louise Mischewski, on behalf of Te Runanga Nui O Te Aupouri, Kaitaia, Northland made two points: 1. They support in principle the release of non-toxic fungal endophytes into the NZ environment, as it reduces the use of pesticides which appears culturally and environmentally sound. 2. They request that Grasslanz monitor the impacts with regards to its introduction, release and performance, with any threats to be reported. Our response to Te Runanga Nui O Te Aupouri – 1. From a commercial viewpoint there will be little enthusiasm to release any technology unless it provides significant benefits. 2. Grasslanz will continue to monitor any technology it releases to the environment for detrimental effects.

Submission from Malcolm Paterson on behalf of Ngāti Whātua Ōrākei expressed the following concerns:

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Application to import for release or to release from containment new organisms

1. That while they recognise the value of the cropping industry to NZ the idea of introducing new endophytes not presently known in the NZ environment to support a non-native plant to flourish is viewed as problematic. 2. The information provided did not directly address matters such as the effect these endophytes might have on non-crop grasses - some of which are weed species in NZ and how these might then compete with native plants. Will these endophytes also encourage weed grasses to flourish? 3. That while the information provided suggests the endophytes cannot spread from plant to plant their understanding of these technicalities is very limited in the absence of previous consideration of this issue and the limited information provided. They would advocate for a precautionary approach that first and foremost values our native ecology, biodiversity and species. Our response to Ngāti Whātua Ōrākei – 1. These endophytes are only able to be used in annual cereal crops and will not be in any contact with land areas dedicated to native species. Cereals as annuals are harvested for food and are therefore unlikely to become weeds. Non-native plants are already a significant part of New Zealand’s economy. Cereals have been in New Zealand for over a century and have not become weeds and as such there is no expectation that these endophytic cereals will become weeds. Infection of cereals with endophytes will reduce farmers’ reliance on pesticides, and will lower economic and environmental costs of production. 2. These endophytes complete their whole life cycle in the grass plant and are incapable of horizontal (between plants) transfer. They cannot move from plant to plant, species to species, or genera to genera. It is likely that the competitive advantage of cereals may be improved by these fungal endophytes but this will result in the suppression of weed species. It is hard to envisage how endophytes in cereals would allow weed grasses to flourish. Cereals are grown in cultivated areas and are not going to encroach on areas of native vegetation. 3. There is considerable evidence to show that moving these endophytes across species is very challenging and technically difficult. There is no chance for this to happen spontaneously. Closely related species of these Neotyphodium endophytes (i.e. other members of the Neotyphodium genus) have been part of New Zealand’s ryegrass pastures for more than 100 years. During that time there has been no documented horizontal spread of Neotyphodium endophytes within or between species. They complete their whole life cycle with the plant as shown in the diagram above (Figure 1). A large body of evidence exists describing the biology of Neotyphodium and how they do not spread horizontally (Bacon & Siegel 1988; Clay 1988; Schardl et al. 1994; Chung and Schardl, 1997; Tadych and White 2007). New Zealand ryegrass pastures contain similar types of endophytes and there has been no evidence of any horizontal transfer. Neotyphodium endophytes live entirely within the intercellular spaces of their grass hosts with the endophyte relying entirely on the host plant for dissemination via the seed or through vegetative structures (Philipson & Christey 1986; Schardl et al. 2004). We agree with taking a precautionary approach. We have made our assessment with this in mind and found that no native ecology, biodiversity or species will be adversely affected by these strains. We will be closely monitoring the release of these organisms despite our firm conviction, based on good science both in New Zealand and overseas, that they will not spread from their host plants, enter the environment or change genetically.

Submission from Edward Ellison, Chair of HSNO Committee for Te Runanga Ngāi Tahu was neither in support or opposition to the application but highlights some of the values to Maori for consideration when thinking about the application from a Maori perspective. These issues include whakapapa, kaitiakitanga and rangatiratanga.

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6. Identification and assessment of beneficial (positive) and adverse effects of the new organism(s)

Adverse effects include risks and costs. Beneficial or positive effects are benefits.  Identification involves describing the potential effects that you are aware of (what might happen and how it might happen).  Assessment involves considering the magnitude of the effect and the likelihood or probability of the effect being realised.

Consider the adverse or positive effects in the context of this application on the environment (e.g. could the organism cause any significant displacement of any native species within its natural habitat, cause any significant deterioration of natural habitats or cause significant adverse effect to New Zealand’s inherent genetic diversity, or is the organism likely to cause disease, be parasitic, or become a vector for animal or plant disease?), human health and safety, the relationship of Māori to the environment, the principles of the Treaty of Waitangi, society and the community, the market economy and New Zealand’s international obligations.

Background

New Zealand’s arable sector is currently worth approximately $1billion per annum to the New Zealand economy (information provided by the Foundation for Arable Research (FAR)). The proportion of land devoted to cereal crops has largely been static over the past 10 years with approximately 40,000 hectares in wheat, 75,000 hectares in barley and 6,500 hectares in oats. Despite the static footprint the volume of grain produced nationally has grown nearly 100% over the past 14 years (Figure 2). This increase in crop yield has improved productivity and helped the sector compete more effectively. This is shown by the growing value of the industry, growing exports and a trend towards using cereal crops as feed to boost agricultural production. Further innovation is required to continue improving crop productivity and driving growth in the industry. The purpose of this application is to do so by enhancing farm productivity through the use of non-toxic endophytes.

Figure 2. New Zealand production of wheat, barley and pulses from 1998 to 2010 (data supplied by FAR).

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Benefits

Improving farm productivity through:  reduced costs of applying synthetic chemicals to New Zealand cereal crops  increased cereal crop yields due to lower rates of: o soil nematodes o insect pests o fungal plant diseases

Worldwide crop loss due to animal pests and pathogens has been estimated at 18% and 16% respectively (Oerke 2006). Cereal crops in New Zealand are no different, suffering attack from plant pathogens, insects and other invertebrates. In cereal crops losses are reduced through the regular applications of fungicides and insecticides, accounting for 30 tonnes and 9 tonnes of active ingredient applied each year respectively (Manktelow et al. 2005). The cost synthetic chemicals and their application has been estimated at $22 million per year (data sourced from FAR). We believe that the use of epichloae endophytes in cereal properties will reduce the need for synthetic chemicals. Currently synthetic chemicals are used in conjunction with a range of methods of pest control including the use of pest resistant cultivars, optimizing time of planting and planting with healthy seeds, effective crop rotation, destruction, and/or burial or removal of crop debris (stubble). Synthetic chemical control includes using a variety of pesticides on plants and/or seeds. At times, the simultaneous application of two or more active substances may be required for the control of pests. Epichloae endophytes produce important anti-insect compounds that will help protect cereal crops from some insect pests without the need to apply expensive and toxic synthetic pesticides and fungicides. Not only will the amount of pesticide used be reduced and farm costs decline. Additionally, epichloae endophytes can be used effectively to provide protection against soil nematodes, a pest farmers currently have few viable tools for managing. We have undertaken a number of tests that indicate epichloae endophytes reduce the rate of crop loss from pests. Cereals can be damaged by an array of invertebrate pests. For example, the principal pests of rye corn include, but are not limited to, nematodes; aphids; thrips; wireworms and white grubs; leatherjackets (Tipula spp.); wheat bulb fly (Delia coarctata); leaf miners (Agromyza spp.); frit fly (Oscinella frit); ground beetle (Zabrus tenebrioides); saddle gall midge (Haplodiplosis marginata); cereal leaf beetles (Oulema melanopus, O. gallaeciana); and slugs.

Effect on soil borne nematodes Soil nematodes are a small invertebrate that can harm crops by either feeding on the plant directly or vectoring plant diseases. As the nematodes are found in the soil it is difficult to control them with synthetic chemicals. It is imperative that an effective and economic control for nematodes is developed. There is good evidence to indicate that epichloae endophytes can be part of this solution. Experiments by Dr Michael Wilson at AgResearch have tested the ability of epichloae endophyte to protect Rye (Secale cereal) from root lesion nematodes (Pratylenchus spp.). Presence of epichloae endophyte caused a significant reduction in numbers of nematodes per root system (Section 8). Furthermore, international evidence suggests that a loline alkaloid found in epichloae endophyte-infected plants has been shown to be nematicidal to the root lesion nematode Pratylenchus scribneri (Bacetty et al. 2009).

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Effect on insect pests We have tested the ability of epichloae endophyte in cereal crops to reduce the impact of major insect pests. We have used a variety of methods and conducted tests on three different insect pests, including:  choice bioassays using the bird cherry oat aphid (Rhopalosiphum padi) a significant pest of cereal plants because it transmits barley yellow dwarf virus  no choice test using Aceria mites a type of mite which transmits the wheat streak mosaic virus in Australia  no choice feeding trials using the light brown apple moth (Epiphyas postvittana) which can cause considerable damage with the larvae feeding on numerous horticultural crops. Our results all significantly indicated that the epichloae endophytes inhibited the pest as demonstrated in the figures in section 8.. In addition an international literature review conducted by Johnson et al 2013 shows epichloae endophytes having a detrimental effect on a wide range of insect pests including: Argentine stem weevil (Listronotus bonariensis) (Rowan et al. 1990, Popay et al. 1990, Barker et al. 1984, Prestidge and Gallagher 1985, Popay et al. 1995,1999); pasture mealybug (Balanococcus poea) (Pennell et al. 2005); African black beetle (Heteronychus arator) (Ball et al. 1997, Popay and Thom 2009); porina (Wiseana cervinata) (Jensen and Popay 2004, Popay et al 2012); root aphid (Aploneura lentisci) (Popay and Gerard 2007, Hume et al 2007, Popay and Thom 2009); aphid (Rhopalosiphum padi) (Wilkinson et al 2000); Japanese beetle larvae (Popillia japonica) (Popay et al. 2009, Jensen et al. 2009); fall armyworm (Sopodoptera frugiperda) and corn borer (Ostrinia nubilalis) (Riedell et al. 1991); grass grub larvae (Costelytra zealandica) (Popay and Lane 2000); and large milkweed bug (Oncopeltus fasciatus) (Yates et al. 1989).

Effect on plant diseases We have in vitro results to show the impact of these endophytes on cereal fungal pathogens. Many of our cereal endophytes have inhibited the development of a range of pathogenic and saprotrophic fungi. For example a number of endophyte strains have significantly (P≤0.05) inhibited the mycelial growth of Fusarium graminearum and Rhizoctonia solani (Section 8). These two pathogens are the causal agents of Fusarium head blight and bare patch, respectively, both devastating diseases of cereal crops including wheat and barley. These endophytes have the potential to provide protection against many cereal diseases and although no mechanism/s of action has been identified to date to account for this inhibition, antibiosis through the production of unknown secondary metabolites is a likely mechanism.

Conclusion on efficacy Our research and literature reviews have consistently highlighted the ability of epichloae endophytes to reduce the impact of pests and pathogens on cereal crops. These effects have been found in a variety of cereal crops, against multiple pests and pathogens, using different experimental methods. We believe that this ability is further evidenced from research linking the alkaloid metabolites produced by epichloae endophytes and reduced pest performance. There is a strong case for considering these benefits to be significant. Moreover, as the fungus is vertically transmitted via seed, once a population is infected the bio-protection effects will be transmitted to all progeny. This will enable tonnes of seed to be produced with this desirable trait cheaply in a way that can then be deployed commercially in cereal cropping systems. Epichloae endophytes therefore have the potential to provide a low cost, natural and sustainable method of controlling insect pests in commercial cereal crops and we believe that they will significantly improve farm performance by reducing the cost of synthetic chemicals and reducing the impact of soil nematodes, insect pests and plant diseases.

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Improve environmental and human health by  reducing the use of synthetic chemicals on New Zealand cereal crops The use of pesticides may be difficult to control in variable environmental conditions leading to unwanted dispersal of toxic compounds, for example by drift of sprays or by soil leaching. In addition, the pests may develop pesticide resistance for a number of reasons, including improper practice and handling, which can pose a real threat to crop yields. Accordingly the use of epichloae endophytes to reduce our reliance on synthetic chemicals will be beneficial to these efforts. All agrichemicals used commercially in New Zealand for insect pest control on cereals have been identified as hazardous to humans and the environment under the Hazardous Substances and New Organisms Act 1996. In section 8 we include a table of all these agrichemicals showing a breakdown of toxicity and ecotoxicity hazard subclasses. However, the use of many pesticides can be problematic due to the known problems associated with the chemicals frequently used for such purposes. Many pesticides are toxic and can be dangerous to human and animal consumers of treated agricultural crops (Casida and Quistad, 1998). In particular, accumulation of toxic pesticides in humans and animals can lead to serious health issues for individuals, particularly during early development. For example, pesticide exposure has been linked to respiratory disorders, developmental cancers and shown to have lasting effects on the development of mental abilities (Zejda et al. 1993). Improving farm productivity through:  increased cereal crop yields due to improved drought tolerance and water use efficiency Increasing evidence is being obtained to indicate that endophytes in wild relatives of cereals provide improved drought tolerance of the host plant (Zhang and Nan 2007; 2010). Under low water treatment, endophytic plants produced more biomass and had higher values in plant height and tiller numbers, but no influence by the fungus were observed in high water treatment. This observation aligns with demonstration that Neotyphodium endophyte infection can help ameliorate abiotic stress such as drought and there may be a selective advantage for grasses from certain dryland regions e.g. Mediterranean (Belesky et al.,1989; Assuero et al. 2000; Kane 2011). Lolines, one of our target metabolites, have been shown to possibly have a role in adapting the plant to water stress deficit (Nagabhyru et al. 2013). The potential yield improvements are substantial with stand losses in tall fescue during drought periods reported at greater than 50% after removing the endophyte from the grass (Schardl et al. 2004). Concluding comments on Benefits

Use of these Neotyphodium endophytes in cereal crops is likely to reduce the impact of pests and diseases leading to a reduction in the use of pesticides. Farm productivity will increase as a result to a reduction in the $22 million per year cost by arable farmers on purchase and application of fungicides and pesticides. Data indicates that these endophytes will reduce the impact of soil borne nematodes on cereal crops – a currently intractable problem with no economically useful mitigating options. Based on published information it is predicted that they will improve drought tolerance and improve grain yields.

Risks Cause significant adverse effect to New Zealand’s inherent genetic diversity or cause any significant displacement of any native species within its natural habitat  by hybridisation with native New Zealand Neotyphodium fungi  by cereal crops outcompeting native plants  by insect deterring chemicals harming native insects

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Hybridisation with native New Zealand Neotyphodium fungi As an asexual species it is impossible for these strains to hybridise with native New Zealand Neotyphodium fungi.

Cereal crops outcompeting native plants The motivation for using fungal endophytes with cereals is to improve resistance to insects and plant pathogens, and tolerance to drought. This will improve plant performance and competitive ability. However, there is no evidence to suggest that this improvement will increase their competitive advantage to the point where they can outcompete native species. This is because:

 Cereals crops are grown under conditions that provide tilled and fertile soils along with intensive management of weeds, and pests and soil nutrients are annuals grown in cultivated land and there is little contact with native species. Cereals are poor performers in wild environments and are quickly outcompeted  Cereals are also grown as an annual crop, at the end of the season no plants are left remaining in the field

Insect deterring chemicals harming native insects There are already endophytes in New Zealand that produce similar insect deterring chemicals. For example an endophyte found in pasture produce the alkaloids loline and peramine. Given the large scale of pastoral farming (10.1 million hectares) the release of endophytes in cereal crops (121,000 hectares) is not going to substantially increase the pressure on native insects. Native insects affected by peramine and/or loline alkaloids include three major pasture pests; grass grub (Costelytra zealandica); porina (Wiseana spp.); and pasture mealybug (Balanococcus poea). Neither grass grub nor porina are affected by ryegrass infected with AR1 endophyte which produces peramine (Thom, et al. 2012; Popay et al. 2012) but feeding by larvae of both species is reduced by loline alkaloids (Popay & Lane 2000; A.J. Popay unpublished). There is no firm evidence of loline toxicity to these insects (A.J. Popay unpublished). Populations of pasture mealybug are much lower on endophyte-infected ryegrass (Pennell et al. 2005) and tall fescue (Pennell & Ball 1999) producing peramine and/or loline alkaloids. The effect of alkaloids themselves has not been tested on pasture mealybug and the mechanism of response to endophyte (i.e. deterrence or toxicity) has not been investigated. Two non-pest species have been tested for their response to endophyte in ryegrass. The native cutworm (Graphania mutans) was not deterred from feeding by a concentration of 10 ppm peramine in artificial diets (Dymock et al. 1989). There is also some evidence from plant/endophyte experiments that peramine reduces feeding by adults of the native weevil (Niceana cervina) but this did not cause a reduction in fecundity or oviposition (Barratt et al. 2008).

Harm human health that of farm animals or the environment

 by introducing alkaloids that are toxic to: o birds o mammals Certain alkaloids, notably ergovaline, have been shown to deter birds from feeding on grass infected with endophytes (Madej and Clay 1991, Conover and Messmer 1996, Pennell and Rolston 2010). The deterrence is the result of fewer insects for birds to feed on (Pennell and Rolston 2012) as well as avoidance behaviour known as “post-digestion feedback” (Mason and Reidinger 1983) after grazing endophyte infected pasture (Madej and Clay 1991, Conover and Messmer 1996, Conover 2003, Pennell and Rolston 2010). This learned aversion should help protect avian species from the adverse effects of grazing infected grasses if alternate food supplies are available (Conover and Messmer 1996).

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As with the grass endophyte programme which over the last 30 years has developed extensive testing of stock classes (Fletcher 1999), this programme will ensure that testing of feed grain and herbage from endophytic plants will be undertaken before any commercial release of the technology. There are risks for farm animals but we will test all of these during the R&D phase and only commercialise endophyte/cereal associations that are safe or have known acceptable and manageable risks.

All fungal endophyte and cereal host plant combinations will be tested for bioactivity using mice models which are accepted as indicating possible mammalian toxicity. It is also important to note that endophytes already exist in New Zealand and have been used commercially for over a decade. We have reviewed the evidence around the toxicity of each of the four alkaloids produced by these strains of endophytes. We find that there is no evidence to indicate they are toxic to any animals except insects. They therefore pose no risk to food safety or the welfare of livestock. Toxicology of Loline To test toxicity, Finch (2012) performed feeding studies using mice following OECD guideline 407 “Repeated dose oral toxicity in rodents”. Groups of mice were fed diets containing a mixture of lolines for 3 weeks at a dose rate of 415 mg/kg/day total lolines. Over this period mouse bodyweight, food consumption, behaviour, motor coordination, heart rate and blood pressure was measured regularly. At the conclusion of the experimental period all animals were killed and a blood sample collected for haematology and clinical biochemistry. A full necropsy was performed on each animal and tissue and organ samples collected for histopathology. No significant differences were observed in any of the parameters. Further experiments were performed by Dr Sarah Finch, AgResearch who showed that no toxicity was observed with the oral dosing of NANL or NAL in mice even at the limit dose of 2000 mg/kg. This allows them to be classified in health category 6.1E using the NZ HSNO system of classification and in category 5 using the globally harmonized system of classification and labelling of chemicals (GHS). In both classification systems this is the lowest category of health hazard. For NFL, no toxicity was observed at 1000 mg/kg but to date two mice have lived at the limit dose of 2000 mg/kg and one has died. To complete this study, one more survivor at 2000 mg/kg would allow classification in category 5 using the GHS system but two more deaths at this dose rate would result in the classification in category 4 using the GHS system. These results from both sub-chronic and chronic toxicity testing have raised no concerns for food safety or livestock health. Toxicology of peramine Dr Sarah Finch, AgResearch tested oral acute toxicity of peramine as prescribed by OECD guideline 425 “Acute oral toxicity – Up-and-down procedure”. An oral dose of 2000 mg/kg given to mice induced no signs of toxicity. This allows peramine to be classified in category 6.1E on the NZ HSNO scale and category 5 using the globally harmonized system of classification and labelling of chemicals (GHS). This is the lowest category of health risk on each scale. Furthermore, peramine administered to lambs orally at a rate of 40 mg/head/day for 5.5 days and 80 mg/head/day for 1.5 days had no effect on animal health (Pownall et al. 1995). This rate would have been at the upper levels recorded in endophytic grasses. These studies raise no concerns for food safety or farm animals. Toxicology of chanoclavines Neotyphodium strains expressing chanoclavine do not result in livestock toxicity but show a strong resistance to insect pests (Fleetwood 2007). The compound ergovaline is bio-active and a known human toxin (ergot poisoning) but no strains in this application produce ergovaline. It should be noted, however, that chanoclavine, an intermediary compound in the ergovaline pathway is present in these strains. Chanoclavine, however, is devoid of any ergot-like biological activity (Berde and Schild 1978).

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These results raise no concerns for farm animals and from the available large animal data we have no evidence that chanoclavine will cause a food safety issue. Toxicology of terpendole E Lolitrem B the neurotoxin implicated for causing staggers in livestock is not produced by the Neotyphodium endophyte strains that are the subject of this application. However, some structurally related indole-diterpenes are produced, the most common being terpendole E.

Dr Sarah Finch, AgResearch Ruakura tested the tremorgenicity of terpendole E using the mouse bioassay. She found that it was non-tremorgenic at the highest level of testing of 8 mg/kg in contrast to lolitrem B which causes significant tremors at 1 mg/kg. Previous research has also indicated that a compound must have an α-oriented hydroxyl at C-13 for it to cause tremorgenicity (Munday-Finch 1997). The structure of terpendole E (Figure 3) shows it has no OH group in the 13 position so we would not expect that this compound would be capable of producing tremors at any dose rate. We have never found any compound which lacks this OH group to be tremorgenic.

OH

13 N H OH O O H Terpendole E Non-tremorgenic

H

O

13 O N H OH O O Lolitrem B O Tremorgenic Figure 3. Comparison of chemical structures of a tremorgenic alkaloid (lolitrem B) (Gallagher et al. 1984) and a precursor but non-tremorgenic compound (terpendole E) (Tomoda et al. 1995).

Concluding comments on Risks

The risks of releasing these organisms from containment are negligible. The organism belongs to a genus already established in New Zealand as endemic. Additionally the Neotyphodium strains that this application is seeking to have approved have been shown to be non-toxic in contrast to well established populations already present in New Zealand perennial pastures. Beyond this the fungus does not survive independently of its host plant. Furthermore, the Neotyphodium endophytes are asexual and not capable of spreading to other species. Transfer between plants is vertical and another plant can only be infected by using specialised techniques cultures developed in laboratory- based isolation. Finally, the cereal/endophyte association will not displace native plants because they are annuals and will be grown in cultivated land away from native plant habitats.

Meeting the minimum standards Release from containment of these Neotyphodium strains will meet the 5 minimum standards required by the Act:

A. cause any significant displacement of any native species within its natural habitat

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I. The endophytes are not able to hybridise with related New Zealand species II. The endophytes will not harm native insects in their natural habitat B. cause any significant deterioration of natural habitats

I. The benefits of the endophytes are real but only moderate, the chance of them turning a weak cereal crop requiring a high degree of nurturing and cultivation into an invasive species are insignificant C. cause any significant adverse effects on human health and safety;

I. The alkaloids expressed by the Neotyphodium strains of interest have been tested and shown to be non-toxic II. Any Neotyphodium-derived alkaloids that have known mammalian toxins are absent from the endophyte strains in this application D. cause any significant adverse effects to New Zealand’s inherent genetic diversity

I. There are no indications that these endophytes will cause any effects to New Zealand’s inherent genetic diversity E. cause disease, be parasitic, or become a vector for human, animal, or plant disease (unless is the purpose of the import or release) I. These Neotyphodium endophytes are not plant pathogens

We believe that the EPA should approve this application because it has the potential to: 1. Revolutionise cereal cropping in New Zealand through reduced inputs of synthetic chemicals (currently estimated to cost $22m per year) and improved drought tolerance or water use with the aim of assisting the industry to continue to achieve its goal of raising crop yields by 5% per annum. 2. Reduce pesticide and fungicide use and ensure that environmental impacts are lower for arable crop production 3. Provide a viable option to safely manage some intractable pests such as soil-borne nematodes 4. Effectively compliment synergistic biocontrol options for the control of introduced pests 5. Have negligible risks to the environment, native flora and fauna, or farm animals and humans

7. Could your organism(s) undergo rapid assessment?

If your application involves a new organism that is or is contained within a veterinary or human medicine, could your organism undergo rapid assessment (s38I of the HSNO Act)?

Describe the controls you propose to mitigate potential risks (if any). Discuss what controls may be imposed under the ACVM Act (for veterinary medicines) or the Medicines Act (for human medicines).

Discuss if it is highly improbable (after taking into account controls if any):  the doses and routes of administration of the medicine would have significant adverse effects on the health of the public or any valued species; and  the organism could form an undesirable self-sustaining population and have significant adverse effects on the health and safety of the public, any valued species, natural habitats or the environment.

Do not include effects of the medicine or new organism on the person or animal being treated with the medicine.

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N/A

If your application involves a new organism (excluding genetically modified organisms), could your organism undergo rapid assessment (s35 of the HSNO Act)?

Discuss if your organism is an unwanted organism as defined in the Biosecurity Act 1993.

Discuss if it is highly improbable that the organism after release:  could form self-sustaining populations anywhere in New Zealand (taking into account the ease of eradication)  could displace or reduce a valued species  could cause deterioration of natural habitats,  will be disease-causing or be a parasite, or be a vector or reservoir for human, animal, or plant disease  will have adverse effects on human health and safety or the environment. N/A

8. Other information

Add here any further information you wish to include in this application including if there are any ethical considerations that you are aware of in relation to your application.

Soil Borne Nematode Trials Presence of epichloae endophyte caused a significant reduction in numbers of nematodes per root system (Figure 4).

9 F 1,21 = 7.44, P = 0.013 8 7 LSD 6 5

4 3 2

Numbers of Nematodes in Rootsin of Nematodes Numbers 1 . 0 E - E +

Figure 4. Effect of an endophyte (AR3046) on the number of nematodes on the roots of cultivar Rahu rye corn (data from unpublished research by Michael Wilson, AgResearch).

Insect Pest Trials

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Effect on bird cherry oat aphid The aphid Rhopalosiphum padi is a significant pest of cereal plants because it transmits barley yellow dwarf virus. In a choice bioassay using tillers in Petri dishes, numbers of R. padi on Elymus mutabilis infected with AR3050 were similar to numbers on meadow fescue infected with its natural endophyte N. uncinatum and significantly less than the number of aphids on the meadow fescue endophyte-free control (Table 2).

Table 2. Number of R. padi aphids found on tillers of E. mutabilis infected with AR3050 and meadow fescue with (MF E+) and without (MF E-) its natural endophyte N. uncinatum in a choice trial over 3 days.

No. aphids/tiller

Endophyte treatment Day 1 Day 2 Day 3

AR3050 2.8 1 1.5

MF E+ 0 0 0.8

MF E- 5.2 7.5 15.3

P-value 0.003 <0.001 <0.001

The results of this trial show that loline alkaloids produced as a result of the symbiotic association formed between E. mutabilis and AR3050 may deter aphid grazing as seen in pasture grasses (Wilkinson et al. 2000).

Effect on Aceria mites Aceria spp., notably A. tosichella, are mites which transmit the wheat streak mosaic virus in Australia. The mite used in these trials has been identified as of Aceria spp., tentatively as A. tosichella. Because of the lack of E. mutabilis controls without endophyte and the unknown effect of plant genotype on the occurrence of Aceria mites, the effects of loline alkaloids on mites was assessed on six endophyte-infected (E+) and six endophyte-free (E-) meadow fescue plants by Dr Alison Popay, AgResearch, Ruakura Research Centre. Aceria mites were counted on the second and third leaf of each of three tillers of each plant. The results are presented in Table 3. Table 3. Average number of Aceria mites on two leaves of three tillers on six meadow fescue plants with (E+) and without (E-) the endophyte N. uncinatum.

E+ E- SED Significance

Tiller 1 65 417 70.6 <0.001

Tiller 2 101 543 97.5 0.001

Tiller 3 59 403 57.1 <0.001

All 224 1363 134.2 <0.001

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The results of this mite trial suggest that loline alkaloids are responsible for deterring mite grazers as has been shown for loline-producing endophytes in pasture grasses (Wilkinson et al. 2000).

Effect on light brown apple moth This insect has known sensitivity to bioactives produced by endophytes in ryegrass (A.J. Popay unpublished data). It has been used here to indicate the presence of bioactives in E. mutabilis plants infected with AR3046. A significant effect of AR3046 was found when E. mutabilis plant material taken from plants infected with AR3046 was incorporated into an artificial diet and fed to light brown apple moth. The average proportion of larvae which established and commenced feeding within the first 24 hours of placement of neonate larvae on the test diets was significantly less on AR3046 in E. mutabilis than in Elymus without endophyte (Table 4). There was very little feeding on AR3046-infected diets after 48 h and the time to the first moult was considerably delayed as a result. Table 4. Proportion of light brown apple moth that had established on diets after 24 h and commenced feeding after 24 and 48 h, and the average time to the first moult when placed on diets incorporating freeze dried plant material from Elymus without endophyte or infected with the loline-producing endophyte AR3046.

Plant Endophyte Proportion Proportion Feeding Time to 1st Established moult

24 h 24 h 48 h Ln days

Elymus sp. Nil 0.91 0.83 0.87 2.176 Elymus mutabilis AR3046 0.05 0.05 0.00 2.716 SED 0.153 0.102 0.129 0.1079 P <0.001 <0.001 <0.001 <0.001

The results in this moth trial show that AR3046 produces loline alkaloids that are responsible for deterring light brown apple moth grazers.

Plant Disease Trials

Trial results for a number of endophyte strains significantly ((P≤0.05) inhibiting the the mycelial growth of Fusarium graminearum (Fig. 5) and Rhizoctonia solani (Fig. 6).

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Figure 5. Cereal endophyte strains that significantly (P≤0.05) inhibited the mycelial growth of Fusarium graminearum in dual culture (data from unpublished research by Stuart Card, AgResearch).

Figure 6. Cereal endophyte strains that significantly (P≤0.05) inhibited the mycelial growth of Rhizoctonia solani in dual culture (data from unpublished research by Stuart Card, AgResearch).

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Table 5. HSNO Classes For Agrichemicals (Insect Pests) For Use On Cereal Crops in New Zealand (Young 2012) Property Toxicity (Human Risk) Ecotoxicity Class Class 6 Class 9 Subclass 6.1 6.3 6.4 6.5 6.6 6.7 6.8 6.9 9.1 9.2 9.3 9.4 Acutely Skin Eye Sensitation Mutagen Carcinoge Reproductive Target Aquatic Soil Terrestrial Terrestrial Toxic Irritation Irritation n Development Organ Vertebrate Invertebrate Acclaim C B A A B A Caterkill 1000 C A B B A A A A Chlorpyrifos C B A A A B A A Counter 20G A B A A A C Cyhella C B A A A B A Dew 600 D B A A D A A Diazinon EC C A A B A A D A A Diazol D B A A D A A Diazol 800 C A A B A A D A A Diazonyl 60 EC C A A B A A D A A Dimethoate C A A, B B A B A A Dovetail D B B A C B C Endure D B B C C Fyfanon 440EW D B A B A A B A Halex CS C B A A A B A Hortcare D B B A A D A A Diazinon 500 EW Karate Zeon C B A A A B A Lannate L C A B B A A B B A Metarex B B D Nuvos A A B B A A A A Phorate A B A A B A B Piricarb C B A B B A A C Rampage Encaps E B A B Trifon C A B B B A A B A C

Table 6. HSNO Classification Descriptions Subclass Description 6.1A Acutely toxic LD50: up to 5 mg/kg 6.1B Acutely toxic LD50: 5 - 50 mg/kg 6.1C Acutely toxic LD50: 50 - 300 mg/kg May 2012 EPA0160 25

Application to import for release or to release from containment new organisms 6.1D Acutely toxic LD50: 300 - 2000 mq/kg 6.1E Acutely toxic LD50: 2000 <5000 mg/kg 6.3A Irritating to the skin 6.3B Mildly irritating to the skin 6.4A Irritating to the eye 6.5A Respiratory sensitisers 6.5B Contact sensitisers 6.6A Known or presumed human mutagens 6.6B Suspected human mutagens 6.7A Known or presumed human carcinoqens 6.7B Suspected human carcinogens 6.8A Known or presumed human reproductive or developmental toxicants 6.8B Suspected human reproductive or developmental toxicants 6.8C Substances that produce toxic human reproductive or developmental effects on or via lactation 6.9A Toxic to human target organs or systems 6.9B Harmful to human target organs or systems 9.1A Very ecotoxic in the aquatic environment 9.1B Ecotoxic in the aquatic environment 9.1C Harmful in the aquatic environment 9.1D Slightly harmful to the aquatic environment or are otherwise designed for biocidal action 9.2A Very ecotoxic in the soil environment 9.2B Ecotoxic in the soil environment 9.2C Harmful in the soil environment 9.2D Slightly harmful in the soil environment 9.3A Very ecotoxic to terrestrial vertebrates (Animals) 9.3B Ecotoxic to terrestrial vertebrates (Animals) 9.3C Harmful to terrestrial vertebrates (Animals) 9.4A Very ecotoxic to terrestrial invertebrates (Insects) 9.4 B Ecotoxic to terrestrial invertebrates (Insects) 9.4C Harmful to terrestrial invertebrates (Insects)

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9. Appendices(s) and referenced material (if any) and glossary (if required)

GLOSSARY A Aceria spp. A genus of mites that are capable of transmitting viruses A. tosichella A species of Aceria commonly known as wheat curl mite which transmits wheat streak mosaic virus Agromyza spp. A genus of stem mining flies affecting grasses and cereals Alkaloids Naturally occurring chemical compounds containing a nitrogen group (usually basic) Apoplastic Space between cells of plant tissues Aploneura lentisci Root aphid Ascomycetes A sub-group of fungi Ascospores Sexual spores produced by ascomycetes B Balanococcus poea Pasture mealybug Bipolar heterothallic mating system A mating system requiring two distinct sexual genotypes C Chanoclavine Tri-cyclic clavine alkaloid produced by certain fungi Clavicipitaceae A group (family) of fungi Clavines Class of ergot alkaloids produced by certain fungi in the biosynthetic route to lysergic acid Costelytra zealandica Grass grub D Delia coarctata Wheat bulb fly E Echinopogon ovatus A native New Zealand grass Elymus A genus (the largest) of grass tribe Hordeeae (=Triticeae) Elymus mutabilis One of around 150 species of the genus Elymus Endophyte An organism that lives within a plant Epichloae A collective term for Epichloë and Neotyphodium Epichloë A genus of fungus within Clavicipitaceae Epiphyas postvittana Light brown apple moth Ergot alkaloids Class of alkaloids produced by certain fungi possessing a modified ergoline structural skeleton Ergovaline An ergot alkaloid containing a tripeptide attached to the ergoline structural skeleton F Fecundity Potential number of eggs that can be laid by an insect Festuca arundinacea Tall fescue (=L. arundinaceum) Festuca pratensis Meadow fescue Fusarium graminearum A plant pathogenic fungus G Graphania mutans Native cutworm species H Haplodiplosis marginata Saddle gall midge Heteronychus arator African black beetle Hordeum A genus of grass tribe Hordeeae (=Triticeae) I Indole diterpenoids Class of alkaloids produced by certain fungi containing an

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indole to which is attached a diterpenoid to form a 6- membered structural skeleton L Listronotus bonariensis Argentine stem weevil Loline A class of alkaloids produced by certain fungi containing a 1- aminopyrrolizidine with an oxygen atom bridging carbons 2 and 7 Lolitrem B Lolitrem structural skeleton to which an additional isoprene unit has been attached at the right-hand end to form a 9- membered ring structure lolitrems Class of indole diterpenes to which a two further isoprene units have been attached at the left-hand end to form an 8- membered ring structural skeleton L. arundinaceum Tall fescue (=Festuca arundinacea) L. multiflorum Annual ryegrass Lolium perenne Perennial ryegrass Lysergic acids An ergot alkaloids containing a carboxylic acid substituted ergoline structural skeleton M Metabolites The intermediates or products of metabolism Mycelial Of mycelia - a collection of fungal hyphae or filaments N Neotyphodium A genus of fungus within Clavicipitaceae Neotyphodium aotearoae Endophyte. A species within Neotyphodium producing anti- insect loline alkaloids N. coenophialum Endophyte. A species within Neotyphodium found in tall fescue grass N. lolii Endophyte. A species within Neotyphodium found in perennial ryegrass N. occultans Endophyte. A species within Neotyphodium N. uncinatum Endophyte. A species within Neotyphodium found in meadow fescue Nematode Unsegmented cylindrical worms belonging to the phylum Nematoda some of which feed in roots of plants Niceana cervina Weevil O Oncopeltus fasciatus Large milkweed bug Oscinella frit Frit fly Ostrinia nubilalis Corn borer Oulema gallaeciana Cereal leaf beetle Oulema melanopus Cereal leaf beetle Oviposition The process of laying eggs Ovule Small egg containing female productive cells within a seed P Pasture persistence The degree to which pasture can persist under pressure from insects Peramine A pyrrolopyrazine alkaloid produced by certain fungi Phenotypes The observable physical or biochemical characteristics of an organism, as determined by both genetic makeup and environmental influences. Phorbia A genus of stem boring flies that infest cereals Pleiotropic Producing more than one effect Poaceae Grass family of plants including cereals Poa matthewsii Grass endemic to NZ from Poa genus Pooideae A subfamily of Poaceae and includes cereals such as wheat,

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barley, oat, rye and many pasture grasses. Popillia japonica Japanese beetle Pratylenchus scribneri Root lesion nematode Pyrrolizidines A class of compounds, examples of which include alkaloids Pyrrollopyrazine A class of compounds, examples of which include alkaloids R Rhizoctonia solani A pathogenic fungi causing various plant diseases such as collar rot, root rot, damping off and wire stem Rhopalosiphum padi Bird cherry oat aphid Ryegrass staggers Neurological impairment of stock caused by the neurotoxin lolitrem B, an alkaloid metabolite produced by some endophytes S Schedonorus phoenix Tall fescue (=Festuca arundinacea) Secale cereale Rye corn Sopodoptera frugiperda Fall armyworm Spermatia A nonmotile male reproductive cell in some fungi Stomatal conductance The measure of the rate of passage of carbon dioxide entering, or water vapor exiting through the stomata of a leaf Stroma A cushion-like mass of fungal tissue, having spore-bearing structures either embedded in it or on its surface Symbionts An organism in a symbiotic relationship especially the smaller member of a symbiotic pair T Terpendole E A terpenedole produced by certain fungi Terpendoles Derivatives containing the indole diterpene structural skeleton Tipula spp. Common name crane fly; larvae of some species feed and cause damage to roots of grasses and cereals Tremorgenicity Pertaining to or emanating from tremorgen, a group of toxins produced by fungi Triticeae A tribe within the Pooideae subfamily of grasses that includes wheat, barley and rye TUB2 genes Tubulin beta-2 gene W Wiseana cervinata Porina Z Zabrus tenebrioides Ground beetle

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Conover, M.R. (2003). Impact of consuming tall fescue seeds infected with endophytic fungus, Neotyphodium coenophialum, on reproduction of chickens. Theriogenology 59: 1313-1323. Conover, M.R. and Messmer, T.A. (1996). Feeding preferences and changes in mass of Canada Geese grazing endophyte-infected tall fescue. The Condor 98: 859-862. Craven, K. D., J. D. Blankenship, et al. (2001). "Hybrid fungal endophytes symbiotic with the grass Lolium pratense." Sydowia 53(1): 44-73. Cunningham, I. J. (1958). "Non-toxicity to Animals of Ryegrass Endophyte and other Endophytic Fungi of New Zealand Grasses." New Zealand Journal of Agricultural Research: 489-497. Dahlman, D.L.; Siegel, M.R.; Bush, L.P. 1997. Insecticidal activity of N-formylloline. XVIII International Grasslands Congress, Winnipeg, Canada http://www.internationalgrasslands.org/files/igc/publications/1997/1991-1913-1005.pdf Dymock, J.J.; Rowan, D.D.; McGee, I.R. 1989. Effects of endophyte-produced mycotoxins on Argentine stem weevil and the cutworm Graphania mutans. Proceedings of the 5th Australasian Grassland Invertebrate Ecology Conference 35-43. Easton, H. S. (2007). "Grasses and Neotyphodium endophytes: Co-adaptation and adaptive breeding." Euphytica 154(3): 295-306. Easton, H.S. and L.R. Fletcher (2007) "The importance of endophyte in agricultural systems - changing plant and animal productivity." Grassland Research & Practice Series, 13: p. 11-18. Easton, M. J. C., J.P.J. Eerens, L.R. Fletcher, D.E. Hume,R.G. Keogh, G.A. Lane, G.C.M. Latch, C.G.L. Pennell, A.J. Popay,M.P. Rolston, B.L. Sutherland and B.A. Tapper (2001). "Ryegrass endophyte: a New Zealand Grassland success story." Proceedings of the New Zealand Grassland Association 63: 37-46. Easton, H. S., G. C. M. Latch, et al. (2002). "Ryegrass Host Genetic Control of Concentrations of Endophyte- Derived Alkaloids." Crop Science 42(1): 51-57. Elm, A. A. and C. P. West (1995). "Endophyte infection effects on stomatal conductance, osmotic adjustment and drought recovery of tall fescue." New Phytologist 131: 61-67. Faeth, S.H. and K. Saikkonen, (2007) Variability is the nature of the endophyte-grass interaction. Grassland Research and Practice Series 13: p. 37-48. Fleetwood, D. J. (2007). Molecular characterisation of the EAS gene cluster for ergot alkaloid biosynthesis in epichloe endophytes of grasses (Doctoral dissertation, Massey University). Fletcher, L.R. (1999). "Non-toxic" endophytes in ryegrass and their effect on livestock health and production. Grassland Research and Practice Series 7: 133-139. Fletcher, L. R. and I. C. Harvey (1981). "An Association of a Lolium Endophyte with Ryegrass Staggers." New Zealand Veterinary Journal 29(10): 185-186. Finch, S. 2012. Toxicology of lolines. AgResearch Internal Report. Gallagher, R.T., Hawkes, A.D., Steyn, P.S., Vleggaar, R (1984). Tremorgenic neurotoxins from perennial ryegrass causing ryegrass staggers disorder of livestock: structure elucidation of Lolitrem B. J. Chem. Soc., Chem. Commun: 614-616. Gwinn, K.D. and Gavin, A.M. (1992). Relationship between endophyte infestation level of tall fescue seed lots and Rhizoctonia zeae seedling disease. Plant Disease 76:911-914. Hill, N.S., Bouton, J.H., Hiatt III, E.E. and Kittle, B. (2005). Seed Maturity, Germination, and Endophyte Relationships in Tall Fescue. Crop Science 45:859–863 Hume, D.E., Ryan, D.L., Cooper, B.M. and Popay A.J. (2007). Agronomic performance of AR37-infected ryegrass in northern New Zealand. Proceedings of the New Zealand Grassland Association 69: 201–205 Jensen, J.G., and Popay, A.J. (2004). Perennial ryegrass infected with AR37 endophyte reduces survival of Porina larvae. New Zealand Plant Protection 57:323-328 Jensen, J.G., Popay, A.J. and Tapper, B.A. (2009). Argentine stem weevil adults are affected by meadow fescue endophyte and its loline alkaloids. New Zealand Plant Protection 62: 12-18 Johnson, L.J., A.C.M. de Bonth, L Briggs, J.R. Caradus, S.C. Finch, D.J. Fleetwood, D.J., L. R. Fletcher, D. E. Hume, R. D. Johnson, A. J. Popay, B. A. Tapper, W. R. Simpson, C. R. Voisey and S. D. Card (2013). The exploitation of epichloae endophytes for agricultural benefit. Fungal Diversity 60:171– 188 Kane, H.H. (2011). Effects of endophyte infection on drought stress tolerance of Lolium perenne accessions from the Mediterranean region. Environmental and Experimental Botany 71 (3): 337– 344.

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Koulman, A., G. A. Lane, et al. (2007). "Peramine and other fungal alkaloids are exuded in the guttation fluid of endophyte-infected grasses." Phytochemistry 68(3): 355-360. Kuldau, G. and C. Bacon (2008). "Clavicipitaceous endophytes: Their ability to enhance resistance of grasses to multiple stresses." Biological Control 46(1): 57-71. Lane, G.A., M.J. Christensen, and C.O. Miles (2000) Coevolution of fungal endophytes with grasses: the significance of secondary metabolites, in Microbial Endophytes, C.W. Bacon and J.F. White, Editors. Marcel Dekker: New York. p. 341-388. Latch, G. C. M. and M. J. Christensen (1985). "Artificial Infection of Grasses with Endophytes." Annals of Applied Biology 107(1): 17-24. Latipbayeva, G., Guthridge, K.M., Rochfort, S., Forster, J.W. and Spangenberg, G.C. (2010). Antifungal properties of pasture grass fungal endophytes. (eds.), Proceedings of the Joint Meeting of the Mycological Society of America and the International Symposium on Endophytes of Grasses. June 28 – July 1, 2010. Lexington, Kentucky (USA). Leuchtmann, A. and K. Clay (1997). The population biology of grass endophytes. Plant Relationships Part B, Springer: 185-202. Li, C.J., Gao, J.H. and Nan, Z.B. (2007). Interactions of Neotyphodium gansuense, Achnatherum inebrians, and plant-pathogenic fungi. Mycological Research III, 1220-1227. Madej, C.W. and Clay, K. (1991). Avian seed preference and weight loss experiments: The effect of fungal endophyte-infected tall fescue seeds. Oecologia 88(2): 296-302. Malinowski, D. P. and D. P. Belesky (2000). "Adaptations of endophyte-infected cool-season grasses to environmental stresses: Mechanisms of drought and mineral stress tolerance." Crop Science 40(4): 923-940. Malinowski, D.P., D.P. Belesky, and G.C. Lewis (2005) Abiotic stresses in endophytic grasses, in Neotyphodium in Cool-Season Grasses, C.A. Roberts, C.P. West, and D.E. Spiers, Editors. Blackwell: Ames, IA. p. 187-199. Manktelow D, Stevens D, Walker J, Gurnsey S, Park N, Zabkiewicz J, Teulon D, Rahman A 2005. Trends in pesticide use in New Zealand: 2004. Report to the Ministry for the Environment, Project SMF4193. Mason, J.R. and Reidinger Jr., R.F. (1983). Importance of Color for Methiocarb-Induced Food Aversions in Red-Winged Blackbirds. The Journal of Wildlife Management. 47(2): 383-393. Moon, C. D., C. O. Miles, et al. (2002). "The evolutionary origins of three new Neotyphodium endophyte species from grasses indigenous to the Southern Hemisphere." Mycologia 94(4): 694-711. Munday-Finch, S.C. Aspects of the chemistry and toxicology of indole-diterpenoid mycotoxins involved in tremorgenic disorders of livestock. Ph.D., University of Waikato, 1997. Nagabhyru, P., Dinkins, R.D., Wood, C.L., Bacon, C.W. and Schardl, C.L. (2013). Tall fescue endophyte effects on tolerance to water-deficit stress. BMC Plant Biology, 13:127. Neil, J. C. (1940). "The Endophyte of Rye-Grass (Lolium pernne)." New Zealand Journal of Science and Technology: 280-291. Oerke, E.C. (2006). Crop losses to pests. Journal of Agricultural Science, 144, 31–43. Pennell, C.; Ball, O.J.P. 1999. The effects of Neotyphodium endophytes in tall fescue on pasture mealy bug (Balanococcus poae). Proceedings of the Fifty Second New Zealand Plant Protection Conference 259-263. Pennell, C.G.L.; Popay, A.J.; Ball, O.J.-P.; Hume, D.E.; Baird, D.B. 2005. 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. New Zealand Journal of Agricultural Research 48: 329-337. Pennell, C.G.L. and Rolston, M.P. (2010). The potential of specialty endophyte-infected grasses for the aviation industry. Presented to Meeting of the International Bird Strike Committee, Cairns (Australia). Pennell, C.G.L. and Rolston, M.P. (2012). Novel uses of grass endophyte technology. Presented at The 8th International Symposium on Fungal Endophyte of Grasses. 16 August 2012. Lanzhou (China). Philipson, M. N. and M. C. Christey (1986). "The Relationship of Host and Endophyte during Flowering, Seed Formation, and Germination of Lolium perenne." New Zealand Journal of Botany 24: 125-134. Popay, A.J. and S.A. Bonos (2005) Biotic responses in endophytic grasses, in Neotyphodium, in Cool- Season Grasses, C.A. Roberts, C.P. West, and D.E. Spiers, Editors. Blackwell: Ames, IA. p. 163-185.

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Popay, A.J.; Cotching, B.; Moorhead, A.; Ferguson, C.M. 2012. AR37 reduces porina populations and plant damage in the field. Proceedings of the New Zealand Grassland Association 74: 165-169. Popay, A.J. and Gerard, P.J. (2007). Cultivar abd endophyte effects on a root aphid, Aploneura lentisci, in perennial ryegrass. New Zealand Plant Protection 60:223-227 Popay, A.J.; Lane, G.A. 2000. The effect of crude extracts containing loline alkaloids on two New Zealand insect pests. pp. 471-475. In: 4th International Neotyphodium/Grass Interactions Symposium. Eds. Paul, V. H.; Dapprich, P. D. Soest, Germany. Popay, A.J. and Thom, E.R. (2009). Endophyte effects on major insect pests in Waikato dairy pasture. Proceedings of the New Zealand Grassland Association 71: 121-126 Popay AJ, Prestidge RA, Rowan DD, Dymock JJ (1990) The role of Acremonium lolii mycotoxins in insect resistance of perennial ryegrass (Lolium perenne). In: Quisenberry SS, Joost RE (eds) Procceedings of the international symposium on Acremonium/grass interactions. Baton Rouge, pp 44–48 Popay AJ, Hume DE, Mainland RA, Saunders CJ (1995) Field resistance to Argentine stem weevil (Listronotus bonariensis) in different ryegrass cultivars infected with an endophyte deficient in lolitrem B. N Z J Agric Res 38:519–528 Popay AJ, Hume DE, Baltus JG, Latch GCM, Tapper BA, Lyons TB, Cooper BM, Pennell CG, Eerens JPJ, Marshall SL (1999) Field performance of perennial ryegrass (Lolium perenne) infected with toxin- free fungal endophytes (Neotyphodium spp.). In: Woodfield DR, Matthew C (eds) Ryegrass endophyte: an essential New Zealand symbiosis, vol grassland research & practice series no. 7. pp 113–122 Popay AJ, Tapper BA, Podmore C (2009) Endophyte-infected meadow fescue and loline alkaloids affect Argentine stem weevil larvae. NZ Plant Prot 62:19–27 Pownall DB, Familton AS, Field RJ, Fletcher LR and Lane GA (1995). The effect of peramine ingestion in pen-fed lambs. Proceedings of the NZ Society of Animal production 55:186 Prestidge, R. A., G. M. Barker, et al. (1991). The Economic Cost of Argentine Stem Weevil in Pastures in New Zealand. Proceedings of the 44th N.Z. Weed and Pest Control Conference. Prestidge RA, Gallagher RT (1985) Lolitrem B - a stem weevil toxin isolated from Acremonium-infected ryegrass. In: 38th New Zealand weed and pest control conference. pp 38–40 Prestidge, R. A. and R. T. Gallagher (1988). "Endophyte fungus confers resistance to ryegrass: Argentine stem weevil larval studies." Ecological Entomology 13(4): 429-435. Riedell W, Kieckhefer R, Petroski R, Powell R (1991) Naturally occurring and synthetic loline alkaloid derivatives: insect feeding behavior modification and toxicity. J Entomol Sci 26:122–129 Rowan DD, Dymock JJ, Brimble MA (1990) Effect of fungal metabolite peramine and analogs on feeding development of Argentine stem weevil (Listronotus bonariensis). J Chem Ecol 16:1683–1695 Rowan, D. and G. Latch (1994). "Utilization of endophyte-infected perennial ryegrasses for increased insect resistance." Biotechnology of endophytic fungi of grasses, CRC Press, Boca Raton 12: 169-183. Rudgers, J.A. and K. Clay (2007) Community and ecosystem consequences of endophyte symbiosis with tall fescue. Grassland Research and Practice Series, 13: p. 19-35. Sanderson, K., Dustow, K. and Dixon, H. (2012). Economic Impact Assessment of Arable Production. A report for the Arable Food Council by Business and Economic Research Limited. Schardl, C. L. (1996). "Epichloe Species: Fungal Symbionts of Grasses." Annual review of Phytopathology 34: 109-130. Schardl, C. L. (2010). "The epichloae, symbionts of the grass subfamily Poideae." Annals of the Missouri Botanical Garden 97(4): 646-665. Schardl, C. L. and K. Clay (1997). Evolution of Mutualistic Endophytes from Plant Pathogens. The Mycota, Springer-Verlag Berlin Heidelberg 1997. 5. Schardl, C.L.; Grossman, R.B.; Nagabhyru, P.; Faulkner, J.R.; Mallik, U.P. 2007. Loline alkaloids: Currencies of mutualism. Phytochemistry 68: 980-996 Schardl, C. L., Leuchtmann, A., Tsai, H. F., Collett, M. A., Watt, D. M., & Scott, D. B. (1994). Origin of a fungal symbiont of perennial ryegrass by interspecific hybridization of a mutualist with the ryegrass choke pathogen, Epichloë typhina. Genetics, 136(4), 1307-1317. Schardl, C. L., A. Leuchtmann, et al. (1997). "Coevolution by Common Descent of Fungal Symbionts (Epichloe spp.) and Grass Hosts." Molecular Biology and Evolution 14(2): 133-143.

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8/11/2013

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May 2012 EPA0160