MEMORANDUM

Application APP203846

Applicant Victoria University of Wellington

To Doug Jones, Acting GM, HSNO

Purpose of the Memorandum Key points for consideration of Application APP203846

Date of Advice 16 March 2020

Purpose 1. This memorandum provides information to support your consideration of application APP203846 to develop, by genetic modifications, agathidicida to investigate the biochemical mechanisms that regulate the physiology and behaviour of the pathogen that causes .

2. This memorandum is intended to be read in conjunction with the draft decision.

The application and information for consideration 3. The application APP203846 from Victoria University of Wellington (‘the applicant’), seeks an approval to develop in containment, by genetic modification, strains of P. agathidicida to develop a further understanding of the biology of the organism that may enable the development of novel control strategies for this important plant pathogen that causes kauri dieback disease. 4. A particular focus is around the genes involved in chemotaxis (i.e. host sensing), motility and germination although other genes involved in other fundamental processes (e.g. metabolism, gene regulation, etc.) may also be explored in order to understand the overall biology of the organism.

5. This application to develop a genetically modified organism in containment was received under section 40 of the Hazardous Substances and New Organisms Act 1996 (HSNO Act). Under section 42 of the HSNO Act, the Authority may make a rapid assessment of the adverse effects of developing the organism if it is satisfied that the host organism and the genetic modification specified for the project meet the criteria prescribed in regulations made under section 41 (specifically, the Hazardous Substances and New Organisms (Low-Risk Genetic Modification) Regulations 2003; ‘the Low-Risk Regulations’), the EPA may approve the application and impose such controls providing for each of the matters specified in Schedule 3 as it thinks fit.

6. As per ‘pathway assessment memorandum’ decided under delegated authority by New Organisms Manager, Stephen Cobb on 26 November 2019, the Authority was satisfied that the development met the criteria for a low-risk genetic modification as specified in the Low-Risk Regulations. 7. The application was formally received on 11 March 2020. 8. A rapid assessment of the adverse effects of developing the genetically modified Phytophthora agathidicida can be found in section 3 of the draft decision.

Host organisms to be developed and ethical and Māori considerations 9. The host organism, Phytophthora agathidicida, is well characterised at the species level, and is a Category 2 host organism as per section 7(2) of the Low-Risk regulations. The project is required to be conducted under a minimum PC2 containment. The applicant will conduct the work in their PC2 facility at the Victoria University of Wellington.

10. (or oomycota) are a group of several hundred organisms that include the Phytophthora genus and although fungal–like, they are considered distinct from fungi and more closely related to algae and green plants. They are commonly called ‘water molds’ and are some of the most devastating plant pathogens, causing diseases that include seedling blights, damping- off, root rots, foliar blights and downy mildews. Several prominent diseases caused by oomycetes include late blight of potatoes, downy mildew of grape vine, sudden oak death and root and stem rot of soybean (Fry and Grünwald. 2010). 11. The genus Phytophthora (Greek for ‘plant destroyer’) belongs to the class Oomycota, order and family , a family that contain more than 600 species (Jung et al. 2018, Weir et al. 2015). Species of Phytophthora can be dispersed either in soil, via surface water movement down rows, from rain splash dispersal, by air, or via movement by humans or invertebrate activity (Bradshaw et al. 2020; Jung et al. 2018; Ristaino and Gumpertz. 2000).The current taxonomic classification of P. agathidicida was completed in 2015 following a period of misidentification and interim naming of the species now known as Phytophthora agathidicida (Weir et al. 2015). 12. The identification of P. agathidicida as the causative agent of kauri dieback disease occurred in the early 1970’s on Great Barrier Island and was subsequently discovered in the Northern region of New Zealand in the 2000’s (reviewed in Bradshaw et al. 2020). Although several pathogenic Phytophthora species exist in New Zealand and some have an important impact on the forestry industry (Scott and Williams. 2014, Jung et al. 2018), the detrimental impact of Phytophthora agathidicida on a culturally significant taonga species such a kauri, has increased the profile of this particular Phytophthora species. 13. Phytophthora agathidicida is a soil-borne species and its dispersal occurs mainly by water movement (rain and floods), and via human activity (Bradshaw et al. 2020).

14. The lifecycle of P. agathidicida progresses through several sporulation stages and produces several types of spores. One of these spore types are motile zoospores which can be dispersed in water or water logged soil (Bradshaw et al. 2020). 15. Although other Phytophthora species may be able to be disseminated under conditions of high humidity in rain and mist, there is currently no known aerial transmission of P. agathidicida. Currently the dispersal of P. agathidicida is thought to be largely soil-borne (e,g. on the footwear File Ref APP203846

of hikers and movement of invasive feral animals such as goats and pigs) (Singh et al. 2017, Bradshaw et al. 2020). 16. Although tools for genetic modification have been developed for other Phytophthora species, genetic modification has not been previously described in the P. agathidicida species. For this project, a range of published laboratory techniques that have been developed by others to study a variety of Phytophthora species will be used. Novel approaches will also be tested and/or adapted by the applicant to develop methods that will allow the genetic modification of P. agathidicida. These will include techniques for post-transcriptional gene silencing, the use of the CRISPR/Cas gene-editing system to silence, modify and delete genes, and the insertion of genes into the genome using plasmids delivered using either polyethylene glycol (PEG)-mediated protoplast transformation or zoospore electroporation. Modifications to these techniques may also be required during the optimization process. 17. The P. agathidicida species to be genetically modified in containment produces several spore types including oospores, zoospores, and sporangia as well as mycelia growth (see application, section 2.3). These spores can potentially survive for many years and have been detected in soil for long periods. Some of these spore types are motile in water (e.g. zoospores) and potentially able to be distributed under conditions of high humidity (reviewed in Bradshaw et al. 2020). As the applicant has several years’ experience in managing the containment and culture systems of non- genetically modified P. agathidicida and its various lifecycle stages in their laboratory (Lawrence et al. 2019), the containment facilities are therefore established and have been operationally tested for the containment of this organism. 18. Although P. agathidicida is currently known to infect kauri ( australis) in the New Zealand environment, inoculates of a range of plant species under controlled conditions have suggested a potentially broader host range (Ryder et al. 2016). Currently the host range is undetermined and has been noted as an important gap in the knowledge of the biology of P. agathidicida and the ecology of kauri forests (Bradshaw et al. 2020). Determining and clarifying the host range of P. agathidicida in the New Zealand environment is an area of active ongoing research. The development of new rapid diagnostics test for detection of P. agathidicida will help to clarify the potential host range of this Phytophthora species (Bradshaw et al. 2020; Winkworth et al. 2020).

19. The applicant stated that kauri trees are not present in the immediate vicinity of the containment facility. Therefore, in the unlikely event of an escape of the genetically modified P. agathidicida from containment, there would be no kauri plant hosts able to support the organism. However, given that the potential host range is currently undetermined and an area of active research, there is a potential risk that the organism may be able to establish itself on other plant species that may be present in the immediate vicinity of the containment facility.

20. The applicant has previously demonstrated involvement with Māori as evinced by a recent publication in which the applicant involved the use of mātauranga to identify candidate plants that may have an inhibitory effect in aspects of the P. agathidicida life cycle, notably the germination of zoospores (Lawrence et al. 2019).

21. The applicant has also stated that they will not use endangered species, New Zealand native flora or fauna, or humans as sources of genetic material for their genetic modification work.

22. Although the applicant has noted that the work is not expected to affect in any way the relationship of Māori and their culture and traditions with ancestral lands, water, sites, wāhi tapu, valued flora and fauna and other taonga, a continued demonstration of engagement and kōrero with Māori interest in this area was provided in the application (section 5). Both Mr Ian Mitchell (member of Te Uri Taniwha hapū, Ngapuhi iwi) and Mr Chris Pairama (Te Taou, Ngati Whaatua) have indicated support for the application for the development work to be conducted in containment.

23. Kaupapa Kura Taiao (KKT) have indicated that they do not have any significant concerns regarding this application and are comforted by the continued engagement demonstrated by the applicant with Māori groups provided in the application.

Comments from external agencies 24. In accordance with section 58(1)a of the HSNO Act, the Department of Conservation (DOC), and the Ministry of Primary Industries (MPI) were provided with the application and asked for comment. 25. To proactively engage in the review process at a stage when the applicant could address any concerns or issues raised by either agency, the applicant provided the EPA with permission for a draft final application to be provided to both MPI and the DOC to review prior to formal receipt. 26. DOC commented that the proposed research will improve the understanding of P. agathidicida and in the longer term, potentially contribute to the development of tools to help manage kauri die- back disease. They also noted that any risks are well managed by containment and that they support approval of the application.

27. MPI comments were provided to the applicant and the draft final application was adjusted to address these comments prior to formal receipt. 28. The applicant has noted Chief Technical Officer (CTO) approval for working with unmodified P. agathidicida strains in the containment facility specified in the application1. The permission letter from MPI and the Unwanted Organism permission conditions specified under Sections 52 and 53 of the Biosecurity Act 1993 have been provided by the applicant (see the Appendix of the application) following MPI’s recommendation.

29. MPI also requested that they be provided with an opportunity to review the formally received final application. Comments from MPI following their review noted the incorporation of their comments into the final draft, particularly the inclusion of the permission letter from the CTO as an appendix, the adequacy of the containment facility and alignment with the containment measures required under the Biosecurity Act 1993. They also noted their overall support for the research work and its alignment with the Kauri Dieback Management Programme to improve conservation values.

The draft decision 30. We assessed the risks associated with this application as being low. We are satisfied that the proposed project meets the low-risk requirements of the Low-Risk Regulations. 31. We believe that the proposed containment regime (MPI-approved PC2 containment facility at Victoria University of Wellington) will be adequate to contain the genetically modified Phytophthora agathidicida specified in this application as the proposed genetic modifications are Category B genetic modifications therefore, a PC2 containment facility would be appropriate.

32. The applicant has also obtained MPI approval to work with P. agathidicida in their research containment facility as an Unwanted Organism.

1 Phytophthora agathidicida is categorised as a Regulated Pest and a Small-Scale Management Programme Organism in the Unwanted Organisms register File Ref APP203846

Proposed controls 33. The proposed controls are set out in Table 2 of the draft decision. The proposed controls are consistent with the MPI/EPA laboratory containment standards in addition to the AS/NZS 2243.3 (2002) containment standards as required in the Low-Risk Regulations. These controls thus prescribe a set method by which the outcome must be achieved, because this is specified in the standards. 34. We note the Unwanted Organism permission conditions specified under Sections 52 and 53 of the Biosecurity Act 1993 that have been granted by MPI for experimental work with P. agathidicida in the containment facility located at the Victoria University of Wellington, Wellington (ATF1741, IMPACT CV440).

35. Based on the information provided in the application, this memorandum, and the draft decision, we recommend that you approve application APP203846 under sections 40(1) and 42A of the HSNO Act.

16 March 2020

Senior Advisor, New Organisms Date Hazardous Substances and New Organisms

References Bradshaw RE, Bellgard SE, Black A, Burns BR, Gerth, ML, McDougal, RL, Scott, P.M., Waipara, NW, Weir, BS, Williams NM, Winkworth RC, Ashcroft T, Bradley EL, Dijkwel PP, Guo Y, Lacey RF, Mesarich CH, Panda P and Horner IJ. 2020. Phytophthora agathidicida: research progress, cultural perspectives and knowledge gaps in the control and management of kauri dieback in New Zealand. Plant Pathol, 69: 3-16. doi:10.1111/ppa.13104

Fry WE and Grunwald, NJ. 2010. Introduction to Oomycetes. The Plant Health Instructor. DOI:10.1094/PHI-I-2010-1207-01. Available: http://www.apsnet.org/edcenter/intropp/PathogenGroups/Pages/IntroOomycetes.aspx

Jung T, Perez-Sierra A, Duran A, Horta Jung M, Balci Y, Scanu B 2018. Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands. Persoonia 40: 182-220.

Lawrence SA, Burgess EJ, Pairama C, Black A, Patrick WM, Mitchell I, Perry NB, Gerth ML 2019. Mātauranga-guided screening of New Zealand native plants reveals flavonoids from kānuka (Kunzea robusta) with anti-Phytophthora activity. Journal of the Royal Society of New Zealand DOI:10.1080/03036758.2019.1648303

Ristaino JB and Gumpertz ML 2000. New Frontiers in the Study of Dispersal and Spatial Analysis of Epidemics Caused by Species in the Genus Phytophthora. Annu Rev Phytopathol 38: 541- 576.

Ryder JM, Waipara NW and Burns BR 2016. What is the host range of Phytopthora agathidicida in New Zealand? Poster Abstract. New Zealand Plant Protection 69 (2016):320

Scott P and Williams N. 2014. Phytophthora diseases in New Zealand Forests. New Zealand Journal of Forestry 59(2): 14–21

Singh J, Curran-Cournane F, Waipara N., Schwendenmann L., Lear G. 2017 Comparison of methods used to detect the organism responsible for kauri dieback, Phytophthora agathidicida, from soil samples. Auckland Council technical report, TR2017/019

Weir BS, Paderes EP, Anand N, Uchida JY, Pennycook SR, Bellgard SE, Beever RE 2015. A taxonomic revision of Phytophthora Clade 5 including two new species, Phytophthora agathidicida and P. cocois. Phytotaxa, [S.l.], v. 205, n. 1, p. 21–38, apr. 2015. ISSN 1179- 3163. doi:http://dx.doi.org/10.11646/phytotaxa.205.1.2.