APPLICATION FORM Containment
To obtain approval for new organisms in containment
Send to Environmental Protection Authority preferably by email ([email protected]) or alternatively by post (Private Bag 63002, Wellington 6140) Payment must accompany final application; see our fees and charges schedule for details.
Application Number
APP201532
Date
11 April 2014
www.epa.govt.nz 2
Application Form Approval for new organism in containment
Completing this application form
1. This form has been approved under section 40 of the Hazardous Substances and New Organisms (HSNO) Act 1996. It only covers importing, development (production, fermentation or regeneration) or field test of any new organism (including genetically modified organisms (GMOs)) in containment. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency or release), a different form will have to be used. All forms are available on our website. 2. If your application is for a project approval for low-risk GMOs, please use the Containment – GMO Project application form. Low risk genetic modification is defined in the HSNO (Low Risk Genetic Modification) Regulations: http://www.legislation.govt.nz/regulation/public/2003/0152/latest/DLM195215.html. 3. It is recommended that you contact an Advisor at the Environmental Protection Authority (EPA) as early in the application process as possible. An Advisor can assist you with any questions you have during the preparation of your application including providing advice on any consultation requirements. 4. Unless otherwise indicated, all sections of this form must be completed for the application to be formally received and assessed. If a section is not relevant to your application, please provide a comprehensive explanation why this does not apply. If you choose not to provide the specific information, you will need to apply for a waiver under section 59(3)(a)(ii) of the HSNO Act. This can be done by completing the section on the last page of this form. 5. Any extra material that does not fit in the application form must be clearly labelled, cross- referenced, and included with the application form when it is submitted. 6. Please add extra rows/tables where needed. 7. You must sign the final form (the EPA will accept electronically signed forms) and pay the application fee (including GST) unless you are already an approved EPA customer. To be recognised by the EPA as an “approved customer”, you must have submitted more than one application per month over the preceding six months, and have no history of delay in making payments, at the time of presenting an application. 8. Information about application fees is available on the EPA website. 9. All application communications from the EPA will be provided electronically, unless you specifically request otherwise.
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Application Form Approval for new organism in containment
Commercially sensitive information
10. Commercially sensitive information must be included in an appendix to this form and be identified as confidential. If you consider any information to be commercially sensitive, please show this in the relevant section of this form and cross reference to where that information is located in the confidential appendix. 11. Any information you supply to the EPA prior to formal lodgement of your application will not be publicly released. Following formal lodgement of your application any information in the body of this application form and any non-confidential appendices will become publicly available. 12. Once you have formally lodged your application with the EPA, any information you have supplied to the EPA about your application is subject to the Official Information Act 1982 (OIA). If a request is made for the release of information that you consider to be confidential, your view will be considered in a manner consistent with the OIA and with section 57 of the HSNO Act. You may be required to provide further justification for your claim of confidentiality. Definitions
Restricting an organism or substance to a secure location or facility to prevent Containment escape. In respect to genetically modified organisms, this includes field testing and large scale fermentation
Any obligation or restrictions imposed on any new organism, or any person in relation to any new organism, by the HSNO Act or any other Act or any Controls regulations, rules, codes, or other documents made in accordance with the provisions of the HSNO Act or any other Act for the purposes of controlling the adverse effects of that organism on people or the environment
Any organism in which any of the genes or other genetic material: Have been modified by in vitro techniques, or Genetically Modified Are inherited or otherwise derived, through any number of replications, from Organism (GMO) any genes or other genetic material which has been modified by in vitro techniques
A new organism is an organism that is any of the following: An organism belonging to a species that was not present in New Zealand immediately before 29 July 1998; An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar prescribed as a risk species, where that organism was not present in New Zealand at the time of promulgation of the relevant regulation; An organism for which a containment approval has been given under the HSNO Act; New Organism An organism for which a conditional release approval has been given under the HSNO Act; A qualifying organism approved for release with controls under the HSNO Act; A genetically modified organism; An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand; An organism present in New Zealand before 29 July 1998 in contravention of
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Application Form Approval for new organism in containment
the Animals Act 1967 or the Plants Act 1970. This does not apply to the organism known as rabbit haemorrhagic disease virus, or rabbit calicivirus A new organism does not cease to be a new organism because: It is subject to a conditional release approval; or It is a qualifying organism approved for release with controls; or It is an incidentally imported new organism
An individual or collaborative endeavour that is planned to achieve a particular Project aim or research goal
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Application Form Approval for new organism in containment
1. Applicant details
1.1. Applicant
Company Name: (if applicable) Landcare Research NZ Ltd
Contact Name: Sarah Dodd
Job Title: Scientist
Physical Address: 231 Morrin Road, St Johns, Auckland 1072
Postal Address (provide only if not the same as the physical): Private Bag 92170, Auckland Mail Centre, Auckland 1142.
Phone (office and/or mobile): +6785585530 or 095744100
Fax: 09 5744101
Email: [email protected]
1.2. New Zealand agent or consultant (if applicable)
Company Name:
Contact Name:
Job Title:
Physical Address:
Postal Address (provide only if not the same as the physical):
Phone (office and/or mobile):
Fax:
Email:
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Application Form Approval for new organism in containment
2. Information about the application
2.1. Type of containment activity Tick the box(es) that best describe your application
Application type Type of new organism
☐ GMO Import into containment ☒ Non-GMO ☐ Develop in containment i.e. regeneration, fermentation GMO or genetic modification ☐ Non-GMO
☐ GMO Field test in containment ☐ Non-GMO
2.2. Brief application description Approximately 30 words about what you are applying to do
To import into containment rust and smut plant pathogens to taxonomically identify and to study in planta those determined low risk to New Zealand in order to verify the benefits, host specificity, and non-target risks associated with their use as weed biocontrol agents.
2.3. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it
Landcare Research recently built an accredited containment facility that now makes it possible to safely investigate microbial plant pathogens within a plant, without the risk of escape into the environment. The purpose of this application is to gain permission to import and hold in the new containment facility foreign plant pathogens known as rusts and smuts with potential as weed biocontrol agents. Our research will confirm if these rusts and smuts are suitable for use as weed biocontrol agents, and are sufficiently specific to the target weed host plants to pose a low risk to the commercial industries and native estates of the countries where they are being considered for release. The meaning of ‘sufficiently specific to the target weed host’ being 1) known to only infect the target weed host or 2) known to infect the weed host and close relatives which are either not present in the country of proposed release or not commercially or culturally valued in the release country (i.e. New Zealand and various Pacific nations).
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Application Form Approval for new organism in containment
Unwanted invasive plants (weeds) cost New Zealand and Pacific nations many millions of dollars every year in control costs, lost income and negative environmental impacts. Weed biocontrol offers a safe and sustainable method for controlling many of the most destructive weeds. Prior to the release of a biocontrol agent it is imperative they are proven to be safe and effective to use. This involves assessment of their ability to control the weed host as well as to rule out the likelihood of unwanted non-target impacts. Only agents that can significantly damage their target weed without posing a threat to other commercially or culturally valued plants should ever be released. Landcare Research has over twenty years of biocontrol research experience, and has identified certain taxonomic groups of organisms that are more likely to provide safe and effective weed biocontrol agents. Landcare Research already has approval to import invertebrates into containment for further study (NOC 100005) and can import plant pathogens, under NOC07009. This application is to import into containment rust and smut pathogens for taxonomic identification, and to allow the full and thorough assessment of pathogens identified as being of low-risk as potential weed biocontrol agents in complete containment. If the assessment process confirms their suitability for weed biocontrol in New Zealand, separate approvals for their release from containment will be sought from the Environmental Protection Agency of New Zealand (EPA).
Only rust and smut pathogens observed in the field to be specific for the target weed will be imported into containment for the inital taxonomic studies. In some cases, the rusts or smuts may not be fully identified, or may be new to science and consequently unnamed. In this case, the first step will be to confirm the identity of the isolate, or to identify the closest taxonomic relative, using DNA sequencing technologies. Once an identity has been confirmed only those pathogens determined to be low risk to New Zealand’s commercial industries and native estates based on their known biology (for named species) or the biology of their closest taxonomic relatives (for new species) will then be subjected to in planta experiments in containment to verify the exact level of risk they would pose.
For previously undescribed new species, identifying the closest taxonomic relative will provide information on the likely biology, life cycles and host range of the new species. However, only pathogens determined as having likely narrow host ranges, and with no known close relatives capable of infecting commercially or culturally valued plants in New Zealand, will be further assessed in planta. To properly assess a plant pathogen’s suitability for weed biocontrol, the pathogens will be inoculated into healthy plants, allowed to infect these plants and to propagate to produce next generation infective propagules. These will then be used to infect more healthy plants in order to complete the disease cycle. These studies are crucial to answering questions relating to the pathogen’s suitability for release in New Zealand and the wider Pacific for the purpose of controlling a target weed without causing significant non-target impacts. In particular it will answer questions relating to the pathogen’s disease life cycle in New Zealand and the Pacific, the pathogen’s potential host range, other potential non-target impacts, and their ability to cause disease symptoms capable of debilitating the target weed to ultimately lead to its control. Should any of the pathogens being tested in containment show unwanted non-target activity during the testing process, they will be destroyed immediately along with any materials they came in contact with such as plant pots, potting mix and plant material.
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Application Form Approval for new organism in containment
2.4. Background and aims of application This section is intended to put the new organism(s) in perspective of the wider activities that they will be used in. You may use more technical language but all technical words must be included in a glossary
Landcare Research recently built a containment facility known as the Beever Plant Pathogen Containment facility (Facility) at their Tamaki site. This Facility has made it possible to now safely investigate microbial plant pathogens in planta in New Zealand, without the risk of escape into the environment. The purpose of this application is to gain permission to import and hold in this new containment facility, foreign plant pathogens (i.e. fungi belonging to the biological groups; rusts and smuts) to firstly perform taxonomic studies and identify the isolates and then secondly verify their safety and effectiveness for the biocontrol of invasive weeds in New Zealand and the wider Pacific using in planta studies in containment. The biology of the pathogens to be subjected to in planta studies will be pre-determined from the literature and current knowledge for known species, or will be based on the known biology of the closest relatives for species identified as being new to science. Only those rusts and smuts determined to be ‘low risk’ to New Zealand’s commercial industries, native estates, humans, animals and other valued plants, by being ‘sufficiently specific to a target weed host’ and not having close relatives that are pathogenic on commercially or culturally valued plants will be considered for further in planta studies. The definition of ‘sufficiently specific to the target weed host’ meaning they are known to only infect the target weed host, and in many instances they will only infect certain biotypes of a host species. However, there may be a small number that could also infect close relatives of the target weed, but these will be plants that are either: a) not present in New Zealand or b) of no, or limited, commercial or cultural value to New Zealand.
Moderate to high risk plant pathogens which would pose a risk to industry, the native estate, other plants, animals or humans would not be used in in planta studies as they are unlikely to be suitable for release as biocontrol agents and would therefore not warrant the investment required for an assessment.
Should studies in containment show that an imported pathogen is suitable for weed biocontrol in New Zealand, we will subsequently apply to EPA for permission to release a pure culture of it from the containment facility. Imported plant pathogens that do not prove to be sufficiently host specific to the target weed will be deemed unsuitable for weed biocontrol and will be destroyed along with any materials that have been used in the assessment process.
Weeds have only become a problem to New Zealand in the last 200 years following European colonisation. Since 1769, at least 25,000 exotic species have been introduced to NZ. It is estimated that a new species naturalises every 39 days and it is known that 270 species have become naturalised between 1988 and 2000. We now have more naturalised species (2108) than native species (New Zealand Plant Conservation Network website http://www.nzpcn.org.nz; Duncan and Williams 2002). In a short time the flora of New Zealand has doubled, posing a considerable threat to the native biodiversity of New Zealand and costing the productive sector millions in measures to control them. Biological control may be the only answer for many widespread weed species, especially
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in environmentally sensitive and other areas where the use of herbicides is unacceptable. Successful biocontrol is critically dependent on understanding the biology and non-target impacts of a potential agent before it can be safely released. Until now, such studies on plant pathogen agents had to be conducted overseas by contracted collaborators. By having the ability to conduct such investigations in New Zealand by New Zealand scientists, we can reduce the time taken to conduct the studies and maintain better quality control over the assessments.
Invasive weeds also cause significant economic and environmental losses in many Pacific nations and in many instances biocontrol offers the only economic and sustainable solution to controlling many of these weeds. Landcare Research has over 20 years’ experience in weed biocontrol and has had a number of successes in controlling weeds with biocontrol agents (e.g. introduction of white smut fungus (Entyloma ageratinae) has controlled mist flower in New Zealand). Recently, Landcare Research has extended its skills and knowledge to help its Pacific neighbours to implement weed biocontrol programmes funded by aid programmes such as the NZAid sponsored programme to implement a five year plan for the biocontrol of the eight most important weeds of the Cook Islands. In order to implement responsible weed biocontrol programmes in these countries, as is the requirement in New Zealand, the biology of potential weed biocontrol agents must be fully investigated so informed decisions on their effectiveness and safety can be assessed. Suitable containment facilities, like the one Landcare Research recently built, do not exist in smaller Pacific countries and so we propose to perform assessments of agents for these programmes in the new Landcare Research facility.
The use of plant pathogens as weed biocontrol agents following a full assessment has been shown to be safe and effective with rusts and smuts performing particularly well. In 2004 a survey of all fungal weed biocontrol pathogens released worldwide was conducted that looked at 26 species of fungi from 26 weeds in 15 countries, which were in turn released to control weeds in seven countries. It was found that since the first deliberate release of a plant pathogen for classical biological control of a weed in 1971, there had not been a single case of unpredicted, post-release non-target damage in the field. In fact, the host-range testing results had often proved conservative, with a number of examples of pathogens attacking non-target plants in pre-release tests, but not being recovered from these species in the field. It was proposed that glasshouse based host-range testing is conservative in its estimate of possible non-target impacts because the trials are conducted under ideal conditions (e.g. density of host plants, temperature, humidity, airflow etc) for infection to occur, conditions that very rarely occur simultaneously in nature. Risk assessments based on rigorous host-range testing, combined with a good understanding of the taxonomy, biology, and ecology of the agent, the target weed, and non-target species, are necessary to ensure that the introduction of an exotic pathogen is a safe and environmentally benign method of weed control (Barton 2004, Waipara et al., 2009, Barton 2012).
Within the new Facility the pathogens would be inoculated into both known hosts and related plants, allowed to infect the plants, express disease symptoms (i.e. in the small number of plants where infection has been successful), form infective propagules, which in turn would be allowed to re-infect new host plants in order to complete the disease cycle. Such investigations will answer questions
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relating to their life cycle, host range, non-target impacts and ability to cause disease in the target weed host. In particular, knowledge of the disease cycle is important to determine for rust pathogens as some require more than one host to complete their life cycle. It is important to have answers to these questions in order to make responsible, well informed decisions on their suitability for weed biocontrol as part of the consideration of their release. The plant pathogens to be imported will generally be imported as viable cultures from recognised international culture collections, from experts in the field (including laboratories) or as collections made by expert Landcare Research staff in the country of origin and, where possible, will be identified to the species level prior to importation. The pure cultures will be imported growing on synthetic media (e.g. petri dish or test tube cultures on agar media), as spores harvested off leaves, or growing in planta (e.g. infected cuttings, seeds, leaves or whole plants). As the first step in importing plant pathogens into containment, all will have their identity either confirmed (if known), or determined (if unknown), using DNA based methods, before any in planta work will commence. All isolates to be imported will be at least known to be of the order Puccinales (i.e. rusts) or classes Ustilaginomycetes and Exobasidiomycetes (i.e. smuts) based on their morphology and observed activity in the field.
Most potential biocontrol agents are found during surveys of the target weed in their country of origin and most are host specific. There is a high chance that some collected microbes will be new to science and not yet taxonomically described and named. Where isolates are new to science, they will require additional testing and examination in New Zealand to resolve their taxonomic identity, but in these instances the cultures would first be imported into our PC2+ laboratory for DNA sequence analysis and preliminary taxonomic examination only. Only those species, or isolates whose close relatives are determined from the literature to be sufficiently host specific and low risk, would be considered for in planta experiments. If the in planta host range assessment confirmed a pathogen was sufficiently host specific, the investment of identifying those unnamed isolates to species level and giving a full taxonomic description would be made. Until identified to species level, all unnamed microorganisms will be under strict containment following the processes used for unwanted foreign plant pathogens. For example this would require showering of all personnel prior to leaving the facility.
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3. Information about the new organism(s)
3.1. Name of organism Identify the organism as fully as possible
Non-GMOs - Provide a taxonomic description of the new organism(s).
GMOs – Provide a taxonomic description of the host organism(s) and describe the genetic modification.
Both - 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. Could the organism form an undesirable self-sustaining population? If not, why not? How easily could the new organism be recovered or eradicated if it established an undesirable self- sustaining population?
This application is to import and hold in containment the plant pathogens described below. Only rust and smut pathogens observed in the field to be specific for the target weed will be imported into containment for the inital taxonomic studies. In some cases, the rusts or smuts may not be fully identified, or may be new to science and consequently unnamed. In this case, the first step will be to confirm the identity of the isolate, or to identify the closest taxonomic relative, using DNA sequencing technologies. Once an identity has been confirmed only those pathogens determined to be low risk to New Zealand’s commercial industries and native estates based on their known biology (for named species) or the biology of their closest taxonomic relatives (for new species) will then be subjected to in planta experiments in containment to verify the exact level of risk they would pose. For previously undescribed new species, identifying the closest taxonomic relative will provide information on the likely biology, life cycles and host range of the new species. However, only pathogens determined as having likely narrow host ranges, and with no known close relatives capable of infecting commercially or culturally valued plants in New Zealand, will be further assessed in planta.
The identification of organisms depends on which of the two steps to the research proposed in this application is under consideration:
Step 1) taxonomic identification of all imported cultures of rust and smut fungi using DNA based methods to confirm the identity of known species, or identify unknown cultures. Use the taxonomic identity and known biology associated with it to assess the potential host range risk of an isolate intended for weed biocontrol.
Step 2) in planta non-target risk assessment studies only for those isolates determined as likely low risk based on their taxonomy assessment in step 1.
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Identification of the Organisms: For Taxonomic studies (step 1): Exotic fungal plant pathogens of the order Puccinales, and classes Ustilaginomycetes and Exobasidiomycetes (i.e. rust and smut fungi) found to be assocated with a specific target weed. Fungal cultures will be sourced from dead and live plant material, and whole plants. If the taxonomic identification step identifies a pathogen as being, or as having a close taxonomic relative (i.e. either same genus or same species; depending on which genus is under consideration), pathogenic to commercially or culturally valued plants in New Zealand, the organism will be destroyed along with everything it had been in contact with, and all research on this pathogen will cease
For In planta experiments (step two): Exotic fungal plant pathogens of the order Puccinales, and classes Ustilaginomycetes and Exobasidiomycetes (i.e. smuts and rusts) no higher than risk group 2 (Australian/New Zealand Standard 2243.3:2002)1, for which their taxonomy indicates that they are likely to only infect the target weed host (and/or close relatives of limited value to NZ). Examples of such pathogens are given below and in Appendix 3.
Type of organism (e.g. bacterium, virus, Fungi fungus, plant, animal, animal cell): Taxonomic class, order and family: Kingdom Fungi, Phyla Basidiomycota (Kirk et al., 2008 pages 78-79): 1. Rusts: order Puccinales (previously also known as Uredinales) (Kirk et al., 2008 page 714). 2. Smuts: orders within the two classes Ustilaginomycetes and Exobasidiomycetes (Begerow et al., 2006).
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Describe the biology and main features of Micro-organisms associated with plants (see the organism including if it has inseparable below for more detail). All will be organisms. pathogenic to a specific target weed plant and in some instances to close relatives which are either not present in NZ or of limited value. Most will produce airborne spores which is a requirement for effective biocontrol agents. Where possible they will be imported as pure cultures on clean synthetic media. But for biotrophic micro- organisms such as rust pathogens that can only survive on living plant material, it will be necessary to import them in association with host plant material. The procedures for importing pathogens in planta are outlined in Appendix 8b of the supplied Containment Facility Manual (Appendix 1). The biology and associated risks are outlined below for each fungal group covered in this application.
Describe if the organism has affinities (e.g. Some will have taxonomic relatives in New close taxonomic relationships) with other Zealand. It is highly improbable (though not organisms in New Zealand. impossible) that introduced rust and smut fungi would hybridise with native fungi, or fungi already present in New Zealand See below for more details on this. Could the organism form an undesirable Yes, most will be able to form a self- self-sustaining population? If not, why sustaining population on their target weed not? host plant(s). Since this project is looking for weed biocontrol agents, it is desirable for them to be self-sustaining. We understand a separate HSNO approval would be required for any release from the containment facility. How easily could the new organism be It would only be possible to recover and recovered or eradicated if it established an eradicate a self-sustaining population undesirable self-sustaining population? outside the containment facility if detected early, and by either spraying with broad spectrum fungicides (if known to be effective against the fungus of interest) or destroying infected host plants. Both would
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require careful monitoring of infected sites to ensure eradication had been achieved. However, the controls in place within the containment Facility make the possibility of an escape close to nil. These controls and processes are outlined in detail in Section 7.
GMOs to import: NA
1Definition of risk groups from the Australian/New Zealand Standard 2243.3:2002 Risk group 1: Microorganisms that are unlikely to cause human, plant, or animal disease. Risk group 2: Microorganisms having moderate individual risk, and limited community risk – a pathogen that can cause human, plant, or animal disease, but is unlikely to be a serious hazard to laboratory workers, the community, livestock, or the environment: laboratory exposures may cause infection, but effective treatment and preventative measures are available, and risk of spread is limited.
Biology and ecology of plant pathogen groups covered in this application
Biological control uses one living organism to control another. There are two kinds of biological control: classical and inundative. In classical biological control the pathogen agents are highly specific for their target host and once they are well established there is no need to make further releases as they will generally persist on their own. By comparison, inundative biological control uses large quantities of less host-specific pathogens to create artificial disease epidemics, but these pathogens do not persist for long in the field and need to be reapplied. This is also known as the ‘bioherbicide’ approach given it has a similarity in application strategy to synthetic herbicides.
Of the weed biocontrol pathogen agents, fungal agents used in a classical manner are currently the most frequently used to control environmental weeds in New Zealand and the Pacific due to the fact that they tend to be highly specific for their target weeds and once established, will disperse naturally without need for further intervention. They are therefore highly likely to be used as weed biocontrol agents in New Zealand well into the future. (Jane Barton pers. comm.)
Those that make good classical weed biocontrol agents are highly host specific and produce aerial spores that self-disseminate from an infected host, land on a new host where they germinate and produce specialised hyphal structures that can then penetrate the new host. Once inside, the pathogen’s mycelium typically invades the host tissues, systemically spreading through the host before producing next generation spores that are released and start the disease cycle
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over again. Some may also produce overwintering spores capable of remaining viable for several years in the ground and debris.
Rust and smut fungi are of particular interest for classical weed biocontrol because these fungi tend to be highly specific to a single host plant and their spores are self-disseminating making their release economically viable and the weed biocontrol sustainable over time. In some instances these fungi can be too specific, with certain strains only pathogenic to certain biotypes of the host plant. In these circumstances it is necessary to release a number of different strains of the pathogen (if available) to target all biotypes of the host in the country of interest. In this instance, each strain of a pathogen needs to be assessed for host range and non-target impacts in containment prior to its release.
Examples of fungal weed biocontrol agents relevant to New Zealand include:
Classical biological control agents:
1) Mouse-ear hawkweed rust (Puccinia hieracii var.piloselloidarum) is specific to mouse-ear hawkweed (Pilosella officianarum formerly Hieracium pilosella) and was initially an accidental introduction to New Zealand discovered here in 1995, but later two further strains were deliberately introduced by AgResearch scientists and released in 1998. The first isolate to arrive did have an initial impact on plant biomass of some plants, but not others. It is assumed all three isolates are now established in New Zealand, but their overall impact on H. pilosella is unknown as it has never been officially evaluated (Tim Jenkins pers comm.)
2) Lantana leaf rust (Prospodium tuberculatum) largely restricted to weed host Lantana camara. EPA recently approved this rust for release in New Zealand (NOR100062) but field releases have not yet begun.
3) Lantana blister rust (Puccinia lantanae) is specific to Lantana camara and mostly affects the pink flowered variety. EPA recently approved this rust for released in New Zealand (NOR100061) but field releases have not yet begun.
4) Mist flower smut (Entyloma ageratinae) specific to mist flower (Ageratina riparia) and released in New Zealand prior to the introduction of the HSNO Act 1996. Control of mist flower was achieved within 8 months in wet areas and within 3-8 years in dry areas. Surveys have since confirmed the predicted no non-target impacts expected which was determined through risk assessments similar to those outlined in this application (Barton et al., 2007).
5) Tradescantia white smut fungus (Kordyana sp.) specific for tradescantia (Tradescantia fluminensis). EPA approved this smut for release in New Zealand (APP201362).
6) Chilean needle grass (CNG) rust (Uromyces pencanus) specifc for CNG (Nassella neesiana). EPA approved this rust for release in New Zealand (ERMA200754).
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7) Bridal creeper rust (Puccinia myrsiphylli) specific for bridal creeper (Asparagus asparagoides) was an accidental introduction first noticed in 2005 and believed to have been a self-introduction from Australia via wind-blown spores. This rust has since been ‘de-newed’ by EPA.
8) Blackberry rust (Phragmidium violaceum) specific for some biotypes of the common blackberry (Rubus fruticosus) was first discovered in 1990 near Rakaia and believed to be self-introduced from Australia via wind-blown spores. It is believed additional strains released in Australia specific for the remaining biotypes of R. fruticosus that were resistant to the first strain, will eventually appear in New Zealand.
Inundative biocontrol agents
Unlike classical agents, inundative agents may not be highly specific for their host and hence importation of such strains has been limited with a preference for indigenous strains. The only example of an imported strain was the Phoma clematidina listed below.
1. Old Man’s beard leaf fungus Phoma clematidina released in New Zealand to control Old Man’s Beard (Clematis vitalba) prior to the introduction of the HSNO Act in 1996. The fungus had some impact early on, but has since disappeared from the release sites (unpublished data). There have been no subsequent releases since then. This pathogen was mostly released in small quantities as is done for classical agents and likely explains its poor performance in controlling C. vitalba.
2. Leaf lesion and tip dieback fungus (Fusarium tumidum) was an indigenous strain trialled as a bioherbicide against gorse (Ulex europaeus) and broom (Cytisus scoparius) with limited success.
Assessment of rust and smuts:
RUSTS - Basdiomycota (class Uredinomycetes; order Puccinales – previously also known as Uredinales):
The use of rusts as classical biocontrol agents: history and safety record
Most of the fungi that have been used for classical biocontrol of weeds are rusts (Basidiomycota: Puccinales (previously Uredinales)), which are obligate parasites (Barton 2004). The characteristics that make rusts particularly useful as classical biocontrol agents are their high virulence, efficient short and long-distance dispersal (via dry, air-borne spores) and their high host- specificity. To date none of the rusts (or other fungi) released as classical biocontrol agents for weeds have caused unpredicted damage to a non-target plant (Barton 2004, Waipara et al., 2009, Barton 2012).
Life Cycle of Typical Rust
The term, rust fungus, refers to the yellow- or rust-coloured uredospores, which are the main dispersal phase of the rust fungi. Rusts have very complicated life-cycles that can include up to
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five spore states (Kirk et al., 2008 pages 576-579): pycniospores also known as spermatia (produced in spermogonia), aeciospores (in aecia), urediniospores (in uredinia), teliospores (in telia) and basidiospores (on basidia) (see Fig. 1). A rust fungus may be autoecious, with its life cycle on only one host, or heteroecious, with spermatia and aecia on one host and uredinia and telia on another. Telia produce teliospores which in turn germinate to produce basidiospores (Fig. 1). If basidiospores, and the telia they came from, are able to infect the same host, then the rust is unequivocally autoecious (i.e. only has one host). If instead basidiospores need an alternate host to complete the cycle, then the rust is heteroecious. Heteroecious rusts are normally NOT considered as potential biocontrol agents because host specificity testing would have to include species related to each of the two alternate hosts (Morin et al., 2006b), and that would make the process overly laborious and expensive. One of the first in planta studies will be to confirm the rust under consideration is autoecious by completing its entire life cycle on the target weed host.
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Fig. 1. Spore Types Potentially Formed by a Rust with a Full Life Cycle (Kirk et al., 2008).
Teliospores (on main host, dark brown, 2n long-lived and often survival stage) (n+n)
nuclear fusion (2n) (Diploid stage)
Meiosis
Basidiospores (on main host, sometimes infect alternate host, transparent, short-lived) (1n) (Haploid stage)
Pycniospores (often on alternate host, transparent, very tiny, very short lived) (Spermatia) (1n) fusion with receptive hypha of opposite mating types dikaryotic mycelium formed
Aeciospores (often on alternate host, infect main host, yellow or orange, moderately (n+n) short-lived)
Urediniospores (on main host, infect main host and produce more reddish rust coloured (n+n) urediniospores or teliospores, moderately long lived)
Repeatedly infect host
(n+n) = Dikaryotic (two separate and genetically different nuclei per cell) (2n)= Diploid (2 sets chromosomes in a single nucleus)(1n) = Haploid and monokayrotic (single set chromosomes in a single nucleus per cell)
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Selection of plants for host range tests
The selection of plants to be included in the host range testing of a classical biocontrol agent usually follows the ‘centrifugal phylogenetic’ method first proposed by Wapshere (1974a). Wapshere suggested that decisions regarding whether or not to include particular plants should be made on the basis of 1) phylogenetic relatedness to the target weed and 2) the likelihood that they would be attacked by the particular agent being tested (Barton 2004). The first point is based on the theory that the more closely related plant species are, the more similar are aspects of their morphology and chemistry, and the more likely they are to be acceptable hosts to particular pathogens. The second point is really just to ensure if there is good reason to include a species on a test list (e.g. because it has been reported in the literature to be a host of the potential agent, or it is a particularly desirable species that lives in close association with the target weed) it is included.
Note, in some instances certain strains of rust can be too narrow in their host range requiring multiple strains to be released in order to achieve effective biocontrol of a target weed. For example, in Australia multiple strains of blackberry rust (Phragmidium violaceum) have been released against blackberry (Rubus fruticosus agg.) which is actually a complex of more than 20 species in the same genus (Rubus).. Initially one strain (F15) was released deliberately in 1991, but one or more strains were also introduced, illegally, in the early 1980s. This resulted in useful control of some weedy Rubus species in Australia, but not others. Consequently, a ‘trap garden’ of resistant blackberry taxa was established in France in 1999 (Morin et al., 2006a). This yielded eight ‘new’ Phragmidium violaceum strains thought to have potential for biocontrol in Australia. The eight ‘new’ strains were released in Australia in April 2004 (Morin et al., 2006a). Note that since its original release the rust has only ever been reported on Rubus species, and only those that were shown to be susceptible in host range tests (Louise Morin, CSIRO Australia, pers. comm.).
Hybridization
A hybrid is the viable progeny that results from a cross between organisms belonging to two different, but closely related (genus level or below) taxa (Kirk et al., 2008, page 324). It is unclear how common this process is in nature with fungi but there is good evidence that it has happened (see, for example Newcombe et al., 2000).
One hypothetical risk of introducing a rust pathogen to New Zealand is that it might hybridize with another rust species of the same genus that already occurs here. Two adverse impacts could result from such a scenario 1) the hybrid rust might back-cross with its parent and thus ‘pollute’ the genetic integrity of a native rust, or 2) the hybrid rust might have a different host range to its parents and therefore pose a risk to non-target plants.
Hybrids can hypothetically be formed in two ways in fungi 1) by anastomosis of germ-tubes, appresoria, substomatal vesicles or intercellular hyphae on the main host or 2) by cross fertilisation of spermogonia on an alternate (aecial) host (Spiers & Hopcroft 1994). Therefore, an
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introduced rust fungus could only form hybrids in New Zealand if it were to infect leaves of a plant that were already infected by another rust species of the same genus. One of the first steps taken in any weed biocontrol project is to conduct a survey of the fungi that already occur on the target weed (Fröhlich & Gianotti 2000). If rust fungi were found on the target weed in the survey they would be identified. If they were of the same genus to the pathogen under consideration for biocontrol, obviously additional investigations into the possibility of hybridization would need to be conducted in order to make an assessment on their suitability for weed biocontrol in New Zealand or the Pacific.
In order for hybridization on an alternate host to occur, and for that to lead to an adverse impact, the following events would need to occur: 1) the rust would need to have an alternate host in its country of origin 2) that alternate host would have to grow in New Zealand, and to have a geographic range that overlapped with that of the target weed,3) teliospores of the introduced rust would have to germinate in New Zealand to produce basidiospores, 4) basidiospores of the introduced rust would have to land on the alternate host in sufficient quantities to cause a significant number of spermogonia to form on infected leaves, 5) those exact same leaves would need to also contain spermogonia of another rust species of the same genus (Note: as an example of likelihood, just 2.7% of NZ’s flora are hosts of species within the genus Uromyces, and most of those do not host the aecial stage) 6) cross fertilization would need to occur, and to lead to a viable aeciospores (this would require the two parent fungi to be genetically compatible, e.g. to have the same number of chromosomes, and compatible alleles), 6) those aeciospores would need to land on a susceptible plant (hybrid fungi reported in the literature often require a hybrid host (see (Spiers and Hopcroft 1994)), and 7) finally, the resulting rust hybrid would then need to outcompete native populations to survive, persist and proliferate in the environment to a level where its population could have an impact. It is therefore highly improbable for the introduction of a rust pathogen to cause negative impacts through hybridization.
Evolution of host range
The “risks of increased non target use, host addition, or host switching through evolution” with respect to fungal pathogens used as classical biocontrol agents were reviewed by Barton (2004). It was concluded in that paper that “The fact that pathogens evolve does present risks of increased target use. However, thorough host-range testing should reveal the fundamental host range of each pathogen, and that should make it possible to give accurate predictions of the magnitude of such risks for each specific project” (Barton 2004). Note that the only reported case of a fungal biocontrol agent altering its host range through time is of increased specialisation. The strain of the rust pathogen Puccinia chondrillina Bubák & P. Syd. released in Australia in 1971 against Chondrilla juncea L. was initially able to attack all three forms of the weed there. Over time, it became more specialised and it is now only able to attack one of these, the narrow-leaved form (Cullen 1971, cited in Barton (2004)).
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Contaminants - mycoparasites and endophytes
Rust mycoparasites exist globally, so all rust shipments must be determined to be clean of such contaminants before they can be imported and subsequently released as biocontrol agents. This is relatively easily done by examining the infected plant material under the microscope prior to harvesting spores for importation or preparing infected whole plants for importation. As part of our research we will investigate and assess the likelihood of local rust mycoparasites significantly hampering the activity of the imported rust once released.
Many rusts are biotrophic and cannot survive separated from their host. It is therefore necessary for them to be transported as part of living plant material (often whole plants are required) in order for them to survive the importation process. Note, HSNO approval will be obtained for any plants not already in New Zealand and MPI import permits will be obtained for all plants to be imported (see Appendix 5 for an example). Given plants are known to harbour symptomless endophytes within their tissues, there is a small risk of importing an undesirable contaminant along with the rust pathogen. Endophytic populations mostly consist of harmless saprophytes, but pathogens have also been found in plants (Dodd et al., 2010). However, the risk of accidentally releasing an undesirable endophyte along with the rust is significantly reduced by transferring the pathogen to clean plants collected in New Zealand and destroying the imported plants once in containment. Depending on the level of risk of an unwanted endophyte being present, the pathogen can be passed through more than one host of New Zealand origin prior to release. The procedure for this is outlined in the containment facility manual in Appendix 7b.
In the unlikely event that an endophyte that is either new to New Zealand or unwanted in New Zealand was discovered, the MPI Facility Verifier (as identified in the facility manual) would be informed immediately. All work with the plant and endohphyte would cease while a decision on the best course of action was made. This decision would be made by MPI and EPA if the organism is new to New Zealand. If the endophyte is deemed unwanted in New Zealand the culture, along with all materials associated with it, would be destroyed. If the endophyte is unlikely to cause a significant threat to New Zealand, HSNO approval will be sought to continue the work. The procedure for this is outlined in the containment facility manual Appendix 7b.
Risk of escape from containment
Given rust pathogens produce air borne spores, there is a risk that these could accidentally be carried out of the containment facility on clothing and equipment. Consequently the containment facility has a number of physical containment features and complementing procedures in place which effectively reduce the risk of this happening to nil. These will be discussed in section 7.
Although rusts are well known for their survival and long distance spread (Viljanen-Rollinson & Cromey 2002) the chances of enough rust spores escaping containment and subsequently landing on their compatible host plant to infect it are effectively nil. This is illustrated by the fact that when deliberately releasing rust pathogens for weed biocontrol, large numbers of spores are required along with manipulation of the environment to ensure spore germination (such as adding
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moisture and covering plants in plastic), successful infection and establishment occur. So, for this to happen naturally, basidiospores of the escaped rust would have to land on a compatible host plant in sufficient quantities to cause a significant number of spermogonia to form on infected leaves. This is considered highly unlikely.
Conclusion
Based on what is known about rusts and their biology, the import into containment of rust plant pathogens that are specific to their target weed host is unlikely to cause any significant negative impacts on native or otherwise valued plants and fungi in New Zealand.
SMUTS - Basidiomycota (sub-phylum Ustilaginomycontina)
The smut fungi are a group of about 1,500 species of pathogens that parasitise flowering plants - most commonly the grass family (Gramineae) and the sedge family (Cyperaceae). They are termed smut fungi because they produce a mass of usually black, powdery spores (teliospores) that often develop in place of the grain in cereal crops or in place of other organs such as the anthers of some flowering plants (e.g. the wild plant Silene alba) (Deacon 2005). Taxonomically, the smut fungi do not form a simple monophyletic group, but are dispersed in amongst a number of different orders within the two classes Ustilaginomycetes and Exobasidiomycetes (Begerow et al., 2006). Those within the Ustilaginomycetes contain what were considered the ‘true smuts’ in the genera Ustilago and Sporisorium because they have the full life cycle as depicted in Fig 2. Those that fall within Exobasidiomycetes were previously not considered true smuts because they either don’t produce teliospores (e.g. order Exobasidiales) or they produce their teliospores within the plant’s tissue (order Entylomatales).
The use of smuts in weed biocontrol: history and safety record.
In contrast to rusts, only a small number of smut fungi have been employed in classical weed biocontrol. In New Zealand only two examples are known to date, with a third being evaluated by the biocontrol community;
1) Mist flower smut (Entyloma ageratinae, order Entylomatales), specific to mist flower (Ageratina riparia), was released in New Zealand prior to the introduction of the HSNO Act 1996. Control of mist flower was achieved within 8 months in wet areas and within 3-8 years in dry areas. Surveys have since confirmed no non-target impacts, as predicted from prior risk assessments similar to those outlined in this application (Barton et al., 2004, 2012). Note, E. ageratinae is of the order Entylomatales because it produces its teliospores inside the leaf tissue and the spores are liberated by rupture of old and decaying litter.
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2) Tradescantia white smut fungus (Kordyana sp., order Exobasidiales), specific to tradescantia (Tradescantia fluminensis), was recently EPA approved for release in New Zealand after it was confirmed to be specific to T. fluminensis when tested against a range of potential hosts in Brazil (as cited in Barreto & Evans 1988). Microfungi of the order Exobasidiales, were previously not considered ‘true smuts’ as the teliospore stage was missing from its life cycle (Vanky 2002 page 1). However, recent ultrastructural, morphological and molecular data has placed this order in the class Exobasidiomycetes of the subphylum Ustilaginomycotina.
3) Pampas (Cortaderia jubata) floral smut (Ustilago sp. order Ustilaginales) was recently identified in Ecuador as a potential biocontrol agent for pampas, requiring further assessment of its suitability. Members of the Ustilaginales were previously considered the ‘true smut’ fungi since they produce the characteristic teliospores and sporulate in the reproductive parts of their host (Vanky 2002 page 2).
To date, none of the smuts (or other fungi) released as classical biocontrol agents for weeds have caused unpredicted damage to a non-target plant (Barton 2004, Waipara et al., 2009, Barton 2012).
Life cycle of typical smuts
The smuts get their name from a Germanic word for dirt because of their dark, thick-walled and dust- like teliospores and are, with few exceptions, plant parasitic fungi, mainly on angiosperms, especially on monocots. Once a smut has successfully infected its host, it essentially hijacks the plants' reproductive systems, forming galls which darken and burst, releasing fungal teliospores which infect other plants nearby. Before infection can occur, the smuts need to undergo a successful mating to form dikaryotic hyphae (two haploid cells fuse to form a dikaryon) (Bakkeren et al., 2008). Some smuts sporulate only in the ovaries of the host, but others form spores in other parts of the plant. An overview of a generalised life cycle for the smuts is given in Fig 2.
Some genera of smut fungi are restricted to a certain host plant genus or family. A few genera are pathogenic on members of several host plant families, including monocotyledonous and dicotyledonous ones. Host specificity for smuts is seen more frequently on monocotyledonous plants as opposed to dicots. For example, sixteen smut genera occur only on host plants in Gramineae, including 230 species of Ustilago (Vanky 2002 page 11). Only smut fungi whose taxonomy indicated they would likely be specific to their target host ‘species’, would ever be considered suitable for weed biocontrol and therefore warrant advancement to ‘step two’ of the evaluation (i.e. in planta experiments in the containment facility).
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Figure 2. Generalised life cycle of smut fungi, life cycle completed on a single host
Teliospores (similar biology and function to the teliospores in rusts) (n+n)
nuclear fusion (2n) (Diploid stage)
Meiosis
Basidiospores (similar biology and function to basidiospores in rust) (1n) (Haploid stage)
Yeast like growth (like spermatia in rusts) (1n)
Conjugation (n+n)
Germination
Infective mycelium (n+n)
Infect plant tissues gall formation in reproductive tissues formation of conidiophores
conidia (similar in function to urediniospores in rust) (n+n)
Repeatedly infect host
The smut species have a simpler life cycle than rusts and the dikaryophase is terminated by the teliospore stage (Fig 1 and 2). The main differences between the smuts and rusts are, while many rust fungi require two different and distantly related hosts to complete their life cycle, smut fungi usually complete their life cycle on only one host, which is always a flowering plant. This makes the prediction and testing for potential host range much easier in the assessment process. Another difference between rust and smut fungi is seen in the way that they infect their host plants. Infections from rust fungi are localized to that part of the plant close to where a germinated urediniospore, aeciospore, or teliospore becomes established. Smut fungi, however, spread to infest the entire plant from a single initial infection site, often targeting specific organs and in particular the reproductive organs of a plant.
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Selection of plants for host range tests
The same discussion presented earlier for the rust applies to the smuts.
Hybridization and Evolution of host range
Due to the similarities between the smut fungi and rust fungi life cycles, the same discussion presented earlier for the rust applies to the smuts.
Contaminants- mycoparasites and endophytes
Unlike the rusts, smut fungi can be cultivated on artificial media and can therefore be imported as pure cultures growing on synthetic media. This eliminates the chances of co-introducing mycoparasites and endophytes associated with plant material when importing the pathogen culture.
Risk of escape from containment
As with the rusts, the smuts produce airborne spores and therefore the same discussion around the risks of escape given for the rusts also applies to the smuts.
Conclusion
Based on what is known about smuts and their biology, the import into containment of smut plant pathogens that are specific for their target weed host is unlikely to cause any significant negative impacts on native or otherwise valued plants and fungi in New Zealand. Summary
• Most of the pathogens that have been used for classical biocontrol of weeds world-wide have been rusts, and they have never caused unpredicted non-target damage in the field, as is true for all pathogens employed in weed biocontrol to date.
• Rusts have complicated life-cycles that include up to five different types of spores (spermatia, urediniospores, teliospores, basidiospores, and aeciospores). Smuts have similar, but simpler life cycles.
• A rust may complete its life cycle on only one host, or it can form some spore types on one host and other types on another (not closely related) host. Therefore extensive host range testing is important to establish a rusts life cycle and number of hosts it can infect.
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• Single strains of some fungi (i.e. rusts) can have too narrow a host range restricting activity on single biotypes of the host, therefore requiring multiple strains to control the full range of target weed genetic diversity in New Zealand or the Pacific.
• It is extremely unlikely that the introduction of a plant pathogen would lead to adverse impacts on native fungi through hybridization
• If fungal pathogens were introduced to New Zealand, there is no reason to believe their host range would broaden over time through evolution
• Many rusts are biotrophic and cannot survive separated from their host. It is therefore necessary for them to be transported as part of living plant material (often whole plants are required) in order for them to survive during the importation process. There is consequently a risk of also importing unwanted endophytes and this also will be part of the investigation.
• Rust mycoparasites exist globally, so all strains must be free of mycoparasite contaminants when imported. Research for biocontrol potential needs to include whether local mycoparasites could significantly hamper the activity of the imported rust once released.
• All fungi require high humidity for spore germination which strongly dictates when inoculum can be released and the likelihood of successful establishment.
• To conclude: The importation of target weed host specific fungal plant pathogens into containment in New Zealand for assessing their suitability as weed biocontrol agents is unlikely to cause any significant negative impact on native or otherwise valued plants or fungi because they will be contained in a purpose-built facility with stringent containment measures in place to prevent escape.
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3.2. Regulatory status of the organism
Is the organism that is the subject of this application also the subject of:
An innovative medicine application as defined in section 23A of the Medicines Act 1981?
☐ Yes ☒ No
An innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997?
☐ Yes ☒ No
4. Information about the containment
4.1. For field tests: The nature and method of the field test Describe the nature and method of the field test and the experimental procedures to be used
N/A
4.2. Proposed containment of the new organism(s) (physical and operational)
Describe how you propose to contain the new organism(s) after taking into account its ability to escape from containment (i.e. the possible pathways for escape)
Microorganism containment is achieved by a combination of the strict laboratory operating procedures and physical specifications of the containment facility, which are audited every 12 and 6 months respectively by a qualified MPI Containment Verifier.
Athough the Landcare Research containment facility is the first facility in New Zealand that allows the safe study of in planta interactions of plant associated microbes, such facilities have been in operation in Australia (CSIRO Canberra), the UK (CABI UK) and the USA (USDA) for a number of years now. To date, there have been no escapes from any of these facilities. The New Zealand facility was designed and built in close consultation with the managers of these international facilities, and as such has achieved a level of containment that exceeds Physical Contianment level 2 described in the following standards: AS/NZS 2243.3.2002: Safety in Laboratories part 3:, “Transitional and Containment Facilities for Invertebrates”, 154.03.02 “Facilities for Microorganisms and Cell Cultures 2007a” and the EPA/MPI standard for “Containment Facilities for Plants: 2007”. See below for more details.
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Theoretically, escape of the plant pathogens from containment could occur by: 1. Inadvertently (or deliberately) being carried out of the facility by personnel on contaminated clothing or equipment.
2. In association with air and/or water movement of the airborne or waterborne spores, arthropod vectors, pollen, seeds, etc.
3. During waste disposal
4. In transit during transfer to, or from, the Facility
To address these risks: • Access to the Facility is restricted to trained personnel only. This includes maintenance contractors who must also be trained in containment procedures prior to entering the building. All entrances to the building require authorised swipe card access.
• All in planta work is done in a Facility which has essentially four levels of sealed compartments between the highest containment area (i.e. growth rooms and laboratory) and the outside. The Facility was designed to comply with Physical Containment level 2 (PC2), AS/NZS 2243.3.2002: Safety in Laboratories part 3:, “Transitional and Containment Facilities for Invertebrates”, 154.03.02 “Facilities for Microorganisms and Cell Cultures 2007a” and the EPA/MPI standard for “Containment Facilities for Plants: 2007”with the additional containment features; 1) directional negative air flow within the building leading to the corridor and wet lab areas, 2) HEPA filtration of all air exiting the building, 3) closed air recirculation systems for each of the high level containment growth rooms, 4) heat treatment of all waste water prior to it leaving the building, 5) decontamination of all equipment and clothing leaving the building and 6) the option of showering for personnel prior to leaving the building; These will be explained in more detail below.
• Containment within the facility is based on four levels of sealed compartments. There are four doors separating each growth room and the laboratory from the outside representing four levels of containment. Each level is monitored for the possibility of escape from the previous level and there are procedures outlined in the facility manual should an escape be detected at any level, to prevent the escapee from making it outside. • The Facility is mechanically ventilated where a directional air-flow is maintained by extracting room air initially through a wire mesh designed to capture all potential invertebrate vectors. Each growth room has a closed air recirculation system to ensure there is no cross contamination between rooms and re-circulated air is not released outside unless vented through a HEPA filter which filters the air at the micron level capturing all spores, pollen, dust particles, etc. There are no opening external windows in the facility and all external glass is double glazed, toughened safety glass.
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• The Facility has a negative air pressure gradient that ensures all air flows towards the inner corridor and wet laboratory areas ensuring no spores, pollen, dust or potential invertebrate vectors can escape the facility when doors are opened.
• Entrance into the facility is via an airlock to ensure any airborne spores, pollen and invertebrates are contained within the facility; There is also a light trap inside the air lock to trap any insect that may make it as far as the air lock.
• To ensure spores, pollen, dust and invertebrates are not inadvertently carried out of the facility by personnel, staff are required to wear coveralls, hair covers, shoe covers, coats and gloves while working in the facility. Personnel must remove all coveralls, hair covers, shoe covers, coats and gloves, and wash their hands and fingernails after working with these organisms before they leave the containment facility. There is also the option of personnel showering prior to exiting the facility when exotic pathogens with airborne spores are being subjected to host range assessment in planta studies.. Note, if at any time there is a consignment in the facility that requires showing of personnel prior to exiting the facility, all personnel exiting the facility must shower first regardless of where they were in the facility. • The design of the Facility accommodates decontamination showering of personnel if required by the MPI import permit. Showering will remove any spores, pollen or invertebrates that may be present on personnel prior to their leaving the facility.
• All cultures of imported micro-organisms are stored within the facility in sealed receptacles (e.g. petri dish, culture bottle, vial etc.) in the main laboratory, or in the case of biotrophs, as a living culture within its host plant contained within one of the high containment growth rooms; • Any in planta work will be carried out in one of the high containment plant growth rooms and at the end of an experiment the room will be cleared of all materials, cleaned and decontaminated according to the procedure outlined in the facility manual. • Any sub-culturing onto artificial media and DNA extraction is done in a Class II Biosafety cabinet. Cabinets are decontaminated after use according to international best practice as described in the Containment manual.
• All micro-organisms, biological and organic waste, coveralls, hair covers, shoe covers, coats and gloves are disposed of and/or decontaminated according to the international best practice as described in the Containment manual.
• No viable propagules will be removed from the laboratory unless approved by a MPI biosecurity inspector to be transported to an appropriate containment facility. All organisms moved into/out of the Facility to a remotely located facility will be double packaged and transported from the laboratory in accordance with Packaging Instruction No. 650 of the IATA) Dangerous Goods Regulations. This includes when they are transferred to the Landcare Research Newhook PC2+ laboratory situated in the main campus building.
• All liquid waste, including cleaning waste, flows into a 1000 L holding tank. The tank contents are then heat treated to kill any viable propagules on a weekly basis or more regularly if
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required. Following treatment the waste is cooled to 40ºC, before being discharged to the sanitary sewer (Appendix 9c Sterilised liquid waste disposal).
• All solid waste is autoclaved within the wet laboratory before exiting the facility for disposal.
• Any equipment leaving the facility is decontaminated first following the appropriate procedures outlined in the facility manual.
In the unlikely event of an incident or accident that may lead to an escape, the PC2 transitional and containment Facility has a micro-organism (all organisms within the scope of this application are micro-organisms) release contingency plan (refer to the Facility Manual). This involves notifying the MPI biosecurity inspector as soon as possible and within 24 hours of noticing breach of containment. The MPI biosecurity inspector will then decide on a course of action depending on their assessment of the risk. Procedures to manage spillage of any micro-organism within and outside the facility and for fire, personal decontamination, theft and sabotage are also addressed in the Facility manual.
Establishment of a self-sustaining population and ease of eradication of a particular micro-organism would depend on its pathogenicity and presence of suitable host plants and vectors in New Zealand. Rust pathogens are used regularly in weed biocontrol and their spores are known to travel great distances in suitable air currents. For example bridal creeper rust (Puccinia myrsiphylli) self-introduced to New Zealand via air currents from Australia. However, the micro-organisms to be imported will be held in a high level of containment at all times. Furthermore, the plant pathogens covered in this application are under consideration for release as weed biocontrol agents and, as such, are more likely to be specific to a relatively small number of plant species, and will require specific environmental conditions for transmission and for infection of a plant. These factors significantly reduce the possibility of establishment of a self-sustaining population in the environment.
Eradication of such a species if containment were breached would depend on the level of risk they pose, the micro-organism causing significant detectable symptoms and the availability of a specific and sensitive laboratory test for detection of an infection. If a pathogen was detected outside the Facility, given the host specificity requirement it could likely be eradicated by destroying all at risk host plants.
5. Māori engagement
Discuss any engagement or consultation with Māori undertaken and summarise the outcomes. Please refer to the EPA policy ‘Engaging with Māori for applications to the EPA’ on our website (www.epa.govt.nz) or contact the EPA for advice.
Consultation with local Māori groups (namely Ngati Paoa Whanau Trust Board, Ngai Tai Umupuia Te Waka Totara Trust, Waikato Tainui Te Kauhanganui Inc. and Ngati Whatua o Orakei Corporate Ltd)
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took place during October-November 2012 by letter and included an invitation to visit the new Facility
(see Appendix 2). No issues were raised during consultation.
6. Risks, costs and benefits
Provide information of the risks, costs and benefits of the new organism(s).
These are the positive and adverse effects referred to in the HSNO Act. It is easier to regard risks and costs as being adverse (or negative) and benefits as being positive. In considering risks, cost and benefits, it is important to look at both the likelihood of occurrence (probability) and the potential magnitude of the consequences, and to look at distribution effects (who bears the costs, benefits and risks).
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.
You must fully complete this section referencing supporting material. You will need to provide a description of where the information in the application has been sourced from, e.g. from in-house research, independent research, technical literature, community or other consultation, and provide that information with this application.
The main benefit of importing, holding and investigating micro-organisms to assess their suitability for weed biocontrol within New Zealand is being able to control the quality of the research undertaken on potential biocontrol agents which will lead to:
even safer releases of biological agents to control weeds.
a reduction in the time it takes to release safe and effective biological control agents by years, resulting in significant cost savings.
the opportunity to increase the skills and knowledge of New Zealand scientists.
Until now, studies to assess the potential host range of plant pathogens with potential for weed biocontrol had to be conducted overseas. By conducting this research for New Zealand, in New Zealand, we can finally control the quality of the research and reduce the number of years it takes to complete it, ultimately leading to even safer biological control of the weeds that threaten NZ and the Pacific region. For example, Landcare Research has contracted Dr Alan Wood in South Africa to
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conduct host-range testing of a rust pathogen of boneseed (Chrysanthemoides moilifera monilifera), an invasive shrub in coastal areas. Dr Wood has experienced difficulty in infecting New Zealand- sourced plants due to the difference in climate between the two countries and the apparent acclimatisation of the New Zealand plants. In addition Dr Wood has had other commitments and consequently hasn’t been able to focus fully on the investigations which are now in their 7th year since first initiated. It is predicted that we would have the answers for this potential biocontrol agent in just 2 years if we could conduct the investigations in New Zealand within the climate controlled Beever containment facility. Reducing the length of time taken to undertake research will also result in significant cost savings.
Potential adverse effects on the environment, in particular on ecosystems and their constituent parts could only occur if a micro-organism escaped containment. In the unlikely event that a pathogen under investigation escapes and is able to infect valued plants in New Zealand the possible adverse effects could be:
• Disease and/or loss of native and valued introduced flora; and
• Deterioration of ecosystems if the disease-causing agent could not be eradicated from the environment and caused loss of genetic diversity and health of our native and valued introduced flora.
Nonetheless, the likelihood of this happening is considered close to zero due to the effectiveness of the containment regime, and the low risk of the micro-organisms to be studied. In the unlikely event that containment were breached, it is unlikely that the micro-organisms would become established in New Zealand since most plant pathogens require specific environmental conditions for transmission and for infection of a plant. Many of the specific vectors such as insects are also not present in New Zealand. Furthermore, all of the plant pathogens to be evaluated in planta in the containment Facility will already have been identified as being most likely specific to the target weed or a small number of related plant species of little commercial or cultural significance, and which often have a restricted distribution in New Zealand. Theoretically, those being assessed for weed biocontrol in Pacific countries would pose a low risk to the New Zealand environment given their host specificity and the likelihood that the host wouldn’t exist in New Zealand.
Furthermore, experience has shown us that in order to cause infection in a susceptible host plant and for the infection to be self-sustaining within the host plant species, pathogen inoculum (i.e. spores) need to be present in sufficiently high numbers to cause the initial infection and environmental conditions meet (e.g. temperature and moisture requirements).
In the highly unlikely event that an organism was to escape containment and an adverse effect was to occur to native and/or valued species, there would be likely flow-on effects to the mauri (life essence) of those species and the role of Māori as kaitiaki (guardians/stewards) in the maintenance and management of mauri.
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However, as described earlier, the nature of containment and likelihood of suitable environment conditions are such that the risk of escape and subsequent infection are close to nil.
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7. Alternative methods and potential effects from the transfer of genetic elements This section is for developments of GMOs that take place outdoors and field tests of GMOs only
Discuss if there are alternative methods of achieving the research objective. Discuss whether there could be effects resulting from the transfer of genetic elements to other organisms in or around the site of the development or field test.
N/A
8. Pathway determination and rapid assessment This section is for the imports of GMOs only
Under section 42B of the HSNO Act your application may be eligible for a rapid assessment. The pathway for your application will be determined after its formal receipt, based on the data provided in this application form. If you would like your application to be considered for rapid assessment (as per the criteria below), we require you to complete this section.
8.1. Discuss whether the GMO(s) to be imported fulfil the criteria The criteria are: The host organism(s) are Category 1 or 2 host organisms as per the HSNO (Low Risk Genetic Modification) Regulations The genetic modifications are Category A or B modifications as per the HSNO (Low Risk Genetic Modification) Regulations and the modifications are not listed in the Schedule of these Regulations The minimum containment of the GMO(s) will be as per the HSNO (Low Risk Genetic Modification) Regulations (PC1 or PC2 as per AS/NZS2243.3:2002)
N/A
9. 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.
In addition to this EPA approval, import approvals from MPI will also be required for each individual plant pathogen to be imported, complete with additional pathogen-specific controls to be imposed. An example of such an approval is given in Appendix 5.
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10. Checklist This checklist is to be completed by the applicant
Application Comments/justifications All sections of the application form completed ☒ Yes ☐ No or you have requested an information waiver (If No, please discuss with an under section 59 of the HSNO Act Advisor to enable your application to be further processed)
Confidential data as part of a separate, ☒ Yes ☐ No identified appendix
Supplementary optional information attached:
Copies of additional references ☒ Yes ☐ No
Relevant correspondence ☐ Yes ☒ No
Administration Are you an approved EPA customer? ☐ Yes ☒ No If Yes are you an: Applicant: ☐ Agent: ☐
If you are not an approved customer, payment of fee will be by: Direct credit made to the EPA bank ☒ Yes ☐ No account (preferred method of payment) ☐ Payment to follow Date of direct credit: (sometime in 2012)
Cheque for application fee enclosed ☐ Yes ☐ No ☐ Payment to follow
Electronic, signed copy of application e-mailed ☒ Yes to the EPA
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Signature of applicant or person authorised to sign on behalf of applicant
☐ I am making this application, or am authorised to sign on behalf of the applicant or applicant organisation.
☒ I have completed this application to the best of my ability and, as far as I am aware, the information I have provided in this application form is correct.
Signature Date
Request for information waiver under section 59 of the HSNO Act
I request for the Authority to waive any legislative information requirements (i.e. concerning the ☐ information that has been supplied in my application) that my application does not meet (tick if applicable).
Please list below which section(s) of this form are relevant to the information waiver request:
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Appendices and referenced material (if any) and glossary (if required)
Appendix 1: Landcare Research Beever Plant Pathogen Containment Facility Manual (Confidential)
Appendix 2: Letter of consultation to local Maori
Appendix 3: Assessment of likely host range for un-named species
Appendix 4: References
Appendix 5: Table of weeds with plant pathogen biocontrol agents imported into NZ and currently under consideration for importation into NZ
Appendix 6: MPI permit for the importation of lantana rust
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APPENDIX 1: Landcare Research Beever Plant Pathogen Containment Facility Manual
See attached file. We request that the contents of this manual remain confidential.
APPENDIX 2: Letter of consultation sent to local Maori Landcare Research - Manaaki Whenua 231 Morrin Road St Johns Auckland 1072
30th October 2012
Tēnā koutou My name is Dr Sarah Dodd and I work for the government Crown Research Institute, Landcare Research – Manaaki Whenua. A key function of ours is to develop environmentally friendly methods to control invasive weeds. One of our more successful methods has been to use biological control, where the plant’s natural enemies are used to control invasive weeds. I plan to apply to the Environment Protection Agency for permission to import and study some of these natural enemies (micro-organisms) in our new national Beever Plant Pathogen Containment Facility, the first of its kind in New Zealand. The Containment Facility will allow us to safely conduct research without the risk of escape, and to gain important knowledge about these natural enemies that will assist in our decision as to their viability and safety. In addition, I will also be seeking permission to import some plant diseases from outside NZ that could pose a threat to NZ if they were to arrive here unexpectedly. These will also be housed in the Containment Facility in a safe and fully contained environment. Here they will be studied to better understand how they function so that we will be better prepared to deal with them should the need arise. Recently Landcare Research has started helping countries in the wider Pacific area in the biological control of their weeds, some of which do not exist in New Zealand. I will also be applying for permission to import some of these plants as part of our work in the Containment Facility. As part of this effort I would like to offer you the opportunity to visit us at 231 Morrin Road, to see our new facility, and to talk about the application outlined above. We have set aside 11am on Thursday 22nd and Friday 23rd November for this purpose. If you could call Delwene Pupuo on (09) 5744178 to inform her which day you can come that would be greatly appreciated. We look forward to hearing from you and to meeting you if, and when, you decide to visit us.
Nāku, nā
Dr. Sarah Dodd Plant Pathologist-Scientist Landcare Research | Maanaki Whenua
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Appendix 3: Assessment of likely host range for un-named species.
Plant pathogens used in classical weed biocontrol programmes are mostly found during surveys of the target weeds in their native habitats where the most diverse collection of the plant’s natural enemies are to be found (Wapshere 1974b). Of particular interest to weed biocontrol practitioners are those pathogens and pests that have evolved with the plant over many years and have subsequently become specific for the target host, and possibly close relatives. As these surveys are usually a first for the target weed, they tend to discover pests and pathogens that are new to science, previously undescribed and un-named. The process of describing and naming a new organism is time- consuming and costly, and so typically in weed biocontrol programmes they are not fully described unless they have passed the non-target risk assessment and they are being considered for release from containment. However, given non-target risk assessments are also expensive and time- consuming (especially when conducted in high level containment facilities like the one at Landcare Research), only pathogens pre-determined by their taxonomy as being most likely to be host-specific would ever be considered for in planta assessments in the containment Facility.
Background to host range prediction based on DNA phylogeny:
The classification of rust and smut species has been traditionally based on both morphological and ecological characters, with emphasis on pathogenicity on specific hosts (Vanky 2002 page 11, Cummins & Hiratsuka 2003 pages 30-31, Cai et al., 2011). These groups have proved relatively stable for over 200 years. So it’s not surprising that the recent application of molecular phylogenetic analyses to the rust and smut fungi has mostly supported previous classifications at the genus level. (Maier et. al, 2003, Bauer et. al, 2006; Aime et. al, 2006; Begerow et. al, 2006). The advantage of molecular technologies is that it has provided a greater level of separation at the species level for those previously indistinguishable by morphological characters. For example, a combination of host- specificity, morphology and DNA sequence data of two microcyclic rust species Puccinia melampodii and P. xanthii (Seier et al., 2009) revealed a new morphospecies, P. xanthii var. parthenii- hysterophorae, previously indistinguishable from P. melampodii apart from the fact that it was associated with the host Parthenium hysterophus. This new morphospecies has since been released as a biocontrol agent against P. hysterophus in Australia.
Another example is Karnal bunt of wheat, caused by the smut fungus Tilletia indica, an important pathogen that is absent and unwanted in Australia. ITS sequence data were used to separate it from morphologically similar but non-pathogenic species that can occur as contaminants in consignments of wheat seed (Levy et al., 2001, Pascoe et al., 2005).
One of the best studied models, Microbotryum, has provided another good example. A narrow species criterion based on host use (Zillig 1921; Baker 1947: as quoted in Cai et al., 2011) was contrasted with the morphologically defined species which defined a single species, Microbotryum violaceum, as the pathogen responsible for almost all anther smuts of Caryophyllaceae (Perlin 1996). Population genetics studies (Bucheli et al., 2000) and the use of the Genealogical Concordance Phylogenetic
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Species Recognition (Le Gac et al., 2007) revealed an absence of gene flow and an ancient differentiation between populations of Microbotryum found on different host plants, which were confirmed by ITS phylogenies as distinct species on different hosts (Lutz et al., 2005, 2008).
It is commonly accepted that rust and smut species are each restricted to host plants within a family or narrower plant taxon. There are many examples describing the rust and smut species of a given plant family (Vanky 2006, Vanky & Lutz 2007), or given host genus (Bauer et. al, 1999; Vanky 2003, Vialle et. al, 2013). As an example, the currently recognised 269 species (USDA fungal database) of the smut fungal genus Entyloma are mostly highly host specific (Begerow et al., 2002). Entyloma ageratinae is specific to mist flower (Ageratina riparia) and, under ideal conditions only, Mexican devil weed (Ageratina adenophora) a plant in the same genus (as cited in Morris 1991 and Morin et al., 1997). Other Entyloma species also seem to be limited to a couple of species in the same genus, or close relatives within a family (Begerow et al., 2002). So, if DNA sequence analysis placed a potential agent in the genus Entyloma, you could be reasonably confident that it had a restricted host range.
Similarly, a Puccinia rust found on a grass is likely to be specific to a subset of grasses that are closely related to its main host. Other Puccinia species are known to specialise on other types of plants (other monocots and less frequently dicots). For example, Puccinia junciphila appears specific to reeds in the genus Juncus while Puccinia lageniferae seems to attack members of the daisy family (Pennycook 1989 page 290).
Note, there are several genera of heteroecious rust fungi that, by definition, require two different hosts to complete their life cycle, and these can be from completely different plant families. Heteroecious rusts are normally NOT considered as potential biocontrol agents because host-specificity testing would have to include species related to each of the two alternate hosts (Morin et al., 2006b), and that would make the process overly laborious and expensive.
We propose to use DNA sequence-based taxonomy to assign new and unnamed rust and smut fungi into a genus and species (where possible), and we will use this identity to predict their likely host range. This will allow us to make well informed decisions about whether or not a pathogen is most likely to have a restricted host range and therefore warrants the investment of importing them into high level containment for further research. If the DNA phylogeny were to identify a pathogen as having a close relative pathogenic to commercially or culturally valued plants in New Zealand, it would fail the criteria for biocontrol and would be destroyed along with everything it had been in contact with.. In most instances a potential agent would be rejected if there was an unwanted pathogen in the same genus or sub-species, but there could be exceptions. For example, the rust genus Puccinia which has provided good host-specific weed biocontrol agents in the past also contains the devastating cereal rust pathogen Puccinia graminis. In this scenario, the literature search will be conducted on the host range of the genus to determine the potential agents likely host range. If the literature indicates it will be specific for the target weed at the species or sub-species level, it will then be assessed for non- target impacts and the valued plants of interest will be included on the host-range test plant list to fully assess the level of risk to these plants. If the potential agent is able to infect the valued plants, the
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agent will be rejected for biocontrol and it will be destroyed along with everything it has come in contact with.
We emphasise, all smut and rust fungi to be imported into containment in New Zealand for the purpose of verifying their suitability for weed biocontrol, will be imported into the high level containment facility at Landcare Research Auckland. This plant pathogen containment facility was specifically designed, built and is actively managed to ensure the absolute containment of microbes and plant pathogens that produce airborne spores, with particular emphasis on containing rusts and smuts.
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APPENDIX 4: References
Aime MC, Matheny PB, Henk DA, Frieders EM, Nilsson RH, Piepenbring M, McLaughlin DJ, Szabo LJ, Begerow D, Sampaio JP, Bauer R, Weiß M, Oberwinkler F, Hibbett DS (2006). An Overview of the higher-level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences. Mycologia 98:896-905. Baker HG (1947). Infection of species of Melandrium by Ustilao violacea (Pers.) Fuckel and the transmission of the resultant disease. Annuals of Botany 11, 333–348. Bakkeren, G, Schirawski, J (2008). Sex in smut fungi: Structure, function and evolution of mating-type complexes. Fungal Genetics and Biology, Vol. 45 (1) S15-S21. Barreto RW, Evans HC (1988). "Taxonomy of a fungus introduced into Hawaii for biological control of Ageratina riparia (Eupatorieae; Compositae), with observations on related weed pathogens". Transactions of the British Mycological Society 91 (1): 81–97. Barton J (2004). How good are we at predicting the field host-range of fungal pathogens used for classical biological control of weeds? Biological Control 31, 99-122. Barton J, Fowler SV, Gianotti AF, Winks CJ, de Beurs M, Arnold GC, Forrester G (2007). Successful biological control of mist flower (Ageratina riparia) in New Zealand: agent establishment, impact and benefits to the native flora. Biological Control 40, 370-385. Barton J (2012). Predictability of pathogen host range in classical biological control of weeds: an update. BioControl 57: 289–305. Bauer R, Begerow D, Sampaio JP, Weiß M, Oberwinkler F (2006). The simple-septate basidiomycetes: a synopsis. Mycological Progress 5, 41–66. Bauer R, Vánky K, Begerow D, Oberwinkler F (1999). Ustilaginomycetes on Selaginella. Mycologia 91,475-484. Begerow D, Lutz M, Oberwinker F (2002). Implications of molecular characters for the phylogeny of the genus Entyloma. Mycological Research 106, 1392-1399. Begerow D, Stoll M, Bauer R (2006). A phylogenetic hypothesis of Ustilaginomycotina based on multiple gene analyses and morphological data. Mycologia 98, 906-916. Bucheli E, Gautschi B, Shykoff JA (2000). Host-specific differentiation in the anther smut fungus Microbotryum violaceum as revealed by micosatellites. Journal of Evolutionary Biology 13,188-198. Cai L, Giraud T., Zhang N., Begerow D., Cai G., Shivas R.G. (2011). The evolution of species concepts and species recognition criteria in plant pathogenic fungi. Fungal Diversity 50,121–133. Cummins GB, Hiratsuka Y (2003) Illustrated Genera of Rust Fungi. 3rd ed. APS Press. The American Phytopathological Society. St.Paul, Minnesota pages 30-31 Dodd SL, Ganley R, Bellgard S, Than D (2010). Endophytes associated with Cirsium arvense - a step toward understanding their role in the success/failure of Sclerotinia sclerotiorum as a bioherbicide. Proceedings of the 17th Australasian Weed Conference, pp. 235-238. Deacon (2005). Smut Fungi In: "Fungal Biology", Blackwell Publishing, London. As cited on http://archive.bio.ed.ac.uk/jdeacon/FungalBiology/grassmut.htm Duncan RP, Williams PA (2002). Darwin's naturalization hypothesis challenged. Nature 417: 608- 609. Fröhlich J, Gianotti AF (2000). 'Fungi recovered from Chilean needle grass, Nassella neesiana (Poacae: Stipeae) in New Zealand.' Landcare Research, Auckland, New Zealand. Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008). 'Dictionary of the Fungi.' (10th Edition) (CSIRO Publishing: Collingwood, Victoria, Australia). Le Gac M, Hood ME, Fournier E, Giraud T (2007). Phylogenetic evidence of host-specific cryptic species in the anther smut fungus. Evolution 61:15–26.
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Levy L, Castlebury LA, Carris LM, Meyer RJ, Pimentel G ( 2001) Internal transcribed spacer sequence-based phylogeny and polymerase chain reaction-restriction fragment length polymorphism differentiation of Tellitia walkeri and T. indica. Phytopathology 91, 935-940. Lutz M, Göker M, Piatek M, Kemler M, Begerow D, Oberwinkler F (2005). Anther smuts of Caryophyllaceae: molecular characters indicate host-dependent species delimitation. Mycological Progress 4,225-238. Lutz M, Piatek M, Kemler M, Chlebicki A, Oberwinkler F (2008). Anther smuts of Caryophyllaceae: molecular analyses reveal further new species. Mycological Research 112,1280-1296. Maier W, Begerow D, Weiß M, Oberwinkler F (2003). Phylogeny of the rust fungi: an approach using nuclear large subunit ribosomal DNA sequences. Canadian Journal of Botany 81, 12-23. Morin L, Aveyard R, Batchelor KL, Evans KJ, Hartley D, Jourdan M (2006a). Additional strains of Phragmidium violaceum released for biological control of blackberry. In 'Proceedings of the 15th Australian Weeds Conference'. Adelaide, South Australia. (Eds C Preston, JH Watts and ND Crossman) pp. 565-568. (Weed management society of South Australia Inc.). Morin L, Evans KJ, Sheppard AW (2006b). Selection of pathogen agents in weed biological control: critical issues and peculiarities in relation to arthropod agents. Australian Journal of Entomology 45, 349-365. Morin L, Hill RL, Matayoshi S (1997). Hawai’i’s successful biological control strategy for mist flower (Ageratina riparia) - Can it be transferred to New Zealand? Biocontrol News and Information 18(3), 77N-88N. Morris MJ (1991) The use of plant pathogens for biological weed control in South Africa. Agriculture, Ecosystems and Environment, 37, 239-255 Newcombe G, Stirling B, McDonald S, Bradshaw HDJ (2000). Melampsora x columbiana, a natural hybrid of M. medusae and M. occidentalis. Mycological Research 104, 261-274. Pascoe IG, Priest MJ, Shivas RG, Cunnington JH (2005) Spores of Telletia ehrhartae, a smut of Ehrharta calycina, are common contaminants of Australian wheat grain, and a potential source of confusion with Telltia indica, the cause of Karnal bunt of wheat. Plant Pathology 54, 161-168. Pennycook, S. (1989). Plant diseases recorded in New Zealand Vol. 2. Plant Diseases Division, Department of Scientific and Industrial Research, Auckland, New Zealand. (Page 290) Perlin MH (1996) Pathovars of Formae speziales of Microbotryum violaceum differ in electrophoretic karyotype. International Journal of Plant Science 157, 447-452. Seier MK, Morin L, van der Merwe M, Evans HC, Romero Á (2009). Are the microcyclic rust species Puccinia melampodii and Puccinia xanthii conspecific. Mycological Research 113,1271-1282. Spiers AG, Hopcroft DH (1994). Comparative studies of the poplar rusts Melampsora medusae, M. larici-populina and their interspecific hybrid M. medusae-populina. Mycological Research 98, 889-903. Vanky K (2002). Illustrated Genera of Smut Fungi. Second Edition. The American Phytopathological Society, Minnesota 238pp. (Pages 1, 2 and 11). Vánky K (2003). The smut fungi (ustilaginomycetes) of Sporobolus (Poaceae). Fungal Diversity 14, 205-241. Vánky K (2006). The smut fungi (ustilaginomycetes) of Restionaceae s. lat. Mycol Balc 3, 19-46. Vánky K, Lutz M (2007). Revision of some Thecaphora species (Ustilaginomyctina) on Caryophyllaceae. Mycological Research 111, 1207-1219. Vialle, A., Feau, N., Frey, P., Bernier, L., Hamelin, R. C (2013). Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi. Molecular Phylogenetics and Evolution 66, 628-644. Viljanen-Rollinson SLH, Cromey MG (2002). Pathways of entry and spread of rust pathogens: implications for New Zealand’s Biosecurity. New Zealand Plant Protection 55:42-48.
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Waipara NW, Barton J, Smith LA, Harman HM, Winks CJ, Massey B, Wilkie JP, Gianotti AF, Cripps MG (2009). Safety in New Zealand weed biocontrol: A nationwide pathogen survey for impacts on non-target plants. New Zealand Plant Protection 62, 41-49. Wapshere AJ (1974a). A strategy for evaluating the safety of organisms for biological weed control. Annals of Applied Biology 77, 201-211. Wapshere, AJ (1974b). Host specificity of phytophagous organisms and the evolutionary centres of plant genera and subgenera. Entomophaga 19, 301-309. USDA database (http://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm) Zillig H (1921). Über spezialisierte Formen beim Antherenbrand, Ustilago violacea (Pers.) Tuck. Zentralbl Bakteriol II 53:33–74.
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APPENDIX 4. Table of weeds with pathogen biocontrol agents imported into NZ and currently under consideration for importation into NZ.
See attached file
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APPENDIX 5. Example of MPI permit for importation of weed biocontrol plant pathogen
On live plant material: Lantana rust MPI import permit 2013048633
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