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. May 2012 EPA0160 3 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. May 2012 EPA0160 4 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