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Staff Advice Report

15 May 2020

Advice to the Decision-making Committee to determine the new organism status of Paenibacillus macerans

Application code: APP204012

Application type and sub-type: Statutory determination

Applicant: Ecotabs New Zealand Ltd

Date application received: 01 May 2020

Purpose of the Application: Information to support the consideration of the determination of Paenibacillus macerans

Executive Summary

On 1 May 2020, the Environmental Protection Authority (EPA) formally received an application from Ecotabs New Zealand Ltd requesting a statutory determination of Paenibacillus macerans. The applicant wishes to import biofertilizer products containing this bacterium into New Zealand.

The applicant provided evidence of the presence of P. macerans in New Zealand soil, in addition to information to support its global distribution in various environments around the world.

After reviewing the information provided by the applicant and found in scientific literature, EPA staff recommend the Hazardous Substances and New Organisms (HSNO) Decision-making Committee (the Committee) to determine that P. macerans is not a new organism for the purpose of the HSNO Act.

Purpose of this document

1. This document has been prepared by the EPA staff to advise the Committee of our assessment of application APP204012 submitted under the HSNO Act (the Act). This document discusses information provided in the application and other sources.

2. The decision path for this application can be found in Appendix 1.

The application

3. On 29 April 2020, Ecotabs New Zealand applied to the EPA under section 26 of the HSNO Act seeking a determination on the new organism status of Paenibacillus macerans.

4. The applicant provides evidence that P. macerans naturally occurs in the New Zealand environment and has a ubiquitous global distribution. Therefore, the applicant considers this bacterium to be not new for the purpose of the HSNO Act.

5. Our assessment includes information contained within the application and any other relevant information found in the scientific literature or elsewhere.

Organism description

6. The bacterium P. macerans (Table 1) was isolated for the first time in 1905, by Schardinger, from cereal-based foodstuff. Originally included within the genus Bacillus, Paenibacillus species were reclassified as a separate genus in 1993 based on phylogenetic data from 16S rRNA gene sequences1 (Ash et al. 1993; Teng et al. 2003). 7. Like all the species in the Paenibacillus genus, P. macerans is a gram-positive2, rod-shaped, spore- forming facultative anaerobe3 bacterium (Johansson & Hederstedt 1999; Noskin et al. 2001).

Taxonomic Unit Classification

Order Bacillales

Family Paenibacillaceae

Genus Paenibacillus

Species P. macerans (Schardinger 1905) Ash et al. 1994

Synonyms Bacillus macerans Schardinger 1905 Paenibacillus thermophilus Zhou et al. 2013

Table 1: Taxonomic description of Paenibacillus macerans

8. Bacteria belonging to the genus Paenibacillus are abundant in nature (Noskin et al. 2001). They can be found in a variety of environments such as soil, water, rhizosphere, vegetable matter, forage, insect larvae and clinical samples (Liang & Wang 2015). These bacteria are relevant to

1 16S rRNA gene sequencing is used as a tool to identify bacteria at the species level and assist with differentiating between closely related bacterial species. 2 Gram-positive bacteria have a cell wall composed of a thick layer called peptidologlycan. 3 Anaerobe: a microorganism that is able to, or can only, live in the absence of oxygen.

humans, animals and plants via the production of antimicrobial compounds useful in medicine and agriculture, as well as enzymes that can be used for bioremediation. However, some Paenibacillus species can also be pathogenic and cause food spoilage (Grady et al. 2016).

9. Paenibacillus macerans is known to promote the growth of plants via the increase of nitrogen fixation and phosphorus solubilisation. The bacterium also produces phytohormones and biocidal substances that can neutralize phytopathogens4 and insect herbivores. Therefore, this species can be used as an agent for biofertilizers or biopesticides (Seldin & Dubnau 1985; Grady et al. 2016).

Distribution of Paenibacillus macerans

Evidence regarding the presence of P. macerans in New Zealand

10. The applicant refers to two theses published after 1998 to provide evidence of P. macerans natural occurrence in the New Zealand environment. 11. Paenibacillus macerans was first mentioned in 2002 by Steve A. Wakelin who studied the bacterium as a potential biological control agent for the common root rot of peas (Wakelin et al. 2002). According to Dr Wakelin (appendix Note 1 of the application form), it is highly likely that this bacterium used during his PhD was isolated from the rhizosphere soil surrounding pea plants collected from the Canterbury region before 1998, as his paper was published in 1998 (Wakelin et al. 1998). The applicant consulted Barry Wards (MPI) who concluded that the thesis was a strong evidence to support the presence of P. macerans in New Zealand prior to 29 July 1998 (see email in application). 12. In 2014, Hoda Ghazalibiglar studied potential microbial biocontrol agents for black rot in brassicas5. Paenibacillus macerans was obtained from Lincoln University’s culture collection and had been isolated from the roots of peas grown in New Zealand. However, we could not determine if the bacterium was issued from a different culture than the one used by Dr Wakelin in the 1990s. The applicant contacted Dr Ghazalibiglar who confirmed that the bacterium had been isolated from New Zealand vegetables but could not confirm when (see email in application).

13. The applicant mentions the presence of the enzyme Cyclodextrin glucanotransferase derived from P. macerans in New Zealand, however, the enzyme is not a living organism and therefore this information cannot be used as evidence in aid of a determination as to the presence of the bacterium in the country.

Evidence regarding the globally ubiquitous distribution of P. macerans

14. Species in the genus Paenibacillus are considered ubiquitous and have been found in various environments (Govindasamy et al. 2010; Wenzler et al. 2015). The applicant refers to the type of environments where the bacterium had been isolated from, according to Wikipedia and the Biosafety Clearing-House websites, to cover its global distribution. Furthermore, we found many scientific papers that used P. macerans from various environmental sources in their studies between 1922 and 2014 (Table 2).

15. As summarised in Table 2, P. macerans has been isolated from environmental samples in both North and South America (Brazil, USA, Mexico, Argentina), Asia (India, China, South Korea, Turkey, Greece), Africa (Nigeria) and Europe (Spain, Italy, Denmark, France). Furthermore, the

4 Phytopathogen: an organism parasitic on a plant host. 5 Brassicas: a plant of a genus that includes cabbage, swede, rape, and mustard.

types of habitats where this bacterium is known to grow (decaying vegetables, soils, wastewater, paperboard products, etc.) are present in New Zealand.

16. Moreover, P. macerans has been found in food such as milk, cheese and rice (Table 2) (Hazards 2009). These products have been imported into or produced within New Zealand before 29 July 1998 and after this date.

Table 2: Samples containing Paenibacillus macerans around the world.

Country Sample Year Reference

Brazil Soils 1993 (Oliveira et al. 1993)

USA Roots of wheat and sorghum; 1922 (Porter et al. 1937; Tilden & Hudson (Oregon, decaying vegetables, in soil and 1935 1942; Haahtela et al. 1981; Nebraska, Iowa) water; cellulose-decomposing 1942 Namjoshi et al. 2010) organisms; paperboard 1976 products 1978 1995-97

India Termite guts; cabbage 2012 (Dheeran et al. 2012; Savitha & cultivated soil; contaminated 2014 Raman 2012; Vishnoi et al. 2014) soils

Spain / Italy Italian cheese; silages 1994 (Rodriguez-Jerez et al. 1994; Rossi 2007 & Dellaglio 2007)

South Korea Humans; animal hospital 2003 (Ko et al. 2008; Oh et al. 2018)

Argentina Rice 2010 (Fangio et al. 2010)

Greece Flat sour evaporated milk 1992 (Kalogridou-Vassiliadou 1992)

France Humans (intracranial infection) 1994 (Bert et al. 1995)

Denmark Rhizosphere, root-free soil, 2002 (Mansfeld-Giese et al. 2002; Li et al. root-free sand and washed 2007 2007) sand; cucumber plants

Nigeria soil, wastewater and food 2007 (Ajayi & Fagade 2007) source

Turkey Gut of Coleoptera 2009 (Çolak et al. 2009)

Mexico Arid mangrove ecosystem 2008 (Sahoo & Dhal 2009)

China Milk powder; sediment from a 2012 (Zhou et al. 2012; Sadiq et al. 2016) hot spring

Evaluation against legislative criteria

17. For an organism to be determined as “not new” under section 26 of the Act, the organism must be shown to lie outside the definition of a new organism as defined in section 2A(1) of the Act:

18. A new organism is- a. an organism belonging to a species that was not present in New Zealand immediately before 29 July 1998: b. 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: c. an organism for which a containment approval has been given under this Act: d. an organism for which a conditional release has been given: e. a qualifying organism approved for release with controls: f. a genetically modified organism: g. an organism that belongs to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand.

19. The decision pathway for a section 26 determination is outlined in Appendix 1. 20. Section 2A(1)(a) of the Act states that a new organism must belong to “a species that was not present in New Zealand immediately before 29 July 1998”. We have evaluated the information regarding P. macerans against this criterion. 21. Regarding other criteria listed in section 2A of the Act, P. macerans : o has not been prescribed as a risk species (section 2A(1)(b)); o has not been approved to be held in containment or released with controls (sections 2A(1)(c), (ca) and (cb)); o is not a genetically modified organism (section 2A(1)(d)); and o has not been eradicated from New Zealand (section 2A(1)(e)).

Comments from Agencies

22. In accordance with section 58(1) of the Act, and the Methodology, the Department of Conservation (DOC) and the Ministry for Primary Industries (MPI) were notified and provided with the opportunity to provide further information on the application. 23. DOC stated that ‘the evidence provided by the applicant strongly suggests the presence of P. macerans in the late 1990s. Coupled with the more recent record, P. macerans does not appear to meet the criteria for a new organism for the purposes of the HSNO Act’. 24. MPI made no comments on the application,beyond those initially provided by Drs Wards and Ghazalibiglar and which were submitted with the application.

Effect on New Zealand’s international obligations

25. EPA staff are not aware of any international obligations that may be affected by this determination.

Recommendation

26. Based on the information available, the bacterium appears to be globally ubiquitous and commonly identified in environments that are also found in New Zealand. Therefore, with its identification in New Zealand soil shortly after the Act came into force, and its ubiquitous distribution, we consider that P. macerans is likely to have been present in New Zealand prior 1998 and to have an ongoing presence today.

27. Therefore, there is sufficient weight of evidence to conclude that Paenibacillus macerans was present in New Zealand prior to 29 July 1998 and continuously after that, and should be determined as not new for the purpose of the Act.

References Ajayi AO, Fagade OE 2007. Heat activation and stability of amylases from Bacillus species. African Journal of Biotechnology 6(10). Ash C, Priest FG, Collins MD 1993. Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Antonie van Leeuwenhoek 64(3-4): 253-260. Bert F, Ouahes O, Lambert-Zechovsky N 1995. Brain abscess due to Bacillus macerans following a penetrating periorbital injury. Journal of Clinical Microbiology 33(7): 1950-1953. Çolak F, Atar N, Olgun A 2009. Biosorption of acidic dyes from aqueous solution by Paenibacillus macerans: Kinetic, thermodynamic and equilibrium studies. Chemical Engineering Journal 150(1): 122-130. Dheeran P, Nandhagopal N, Kumar S, Jaiswal YK, Adhikari DK 2012. A novel thermostable xylanase of Paenibacillus macerans IIPSP3 isolated from the termite gut. Journal of Industrial Microbiology & Biotechnology 39(6): 851-860. Fangio MF, Roura SI, Fritz R 2010. Isolation and identification of Bacillus spp. and related genera from different starchy foods. Journal of Food Science 75(4): M218-M221. Govindasamy V, Senthilkumar M, Magheshwaran V, Kumar U, Bose P, Sharma V, Annapurna K 2010. Bacillus and Paenibacillus spp.: potential PGPR for sustainable agriculture. Plant growth and health promoting bacteria, Springer. Pp. 333-364. Grady EN, MacDonald J, Liu L, Richman A, Yuan Z-C 2016. Current knowledge and perspectives of Paenibacillus: a review. Microbial Cell Factories 15(1): 203. Haahtela K, Wartiovaara T, Sundman V, Skujiņš J 1981. Root-associated N2 fixation (acetylene reduction) by Enterobacteriaceae and Azospirillum strains in cold-climate spodosols. Appl. Environ. Microbiol. 41(1): 203-206. Hazards EPoB 2009. Scientific Opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2009 update). EFSA Journal 7(12): 1431. Johansson P, Hederstedt L 1999. Organization of genes for tetrapyrrole biosynthesis in gram-positive bacteria. Microbiology 145(3): 529-538. Kalogridou-Vassiliadou D 1992. Biochemical activities of Bacillus species isolated from flat sour evaporated milk. Journal of Dairy Science 75(10): 2681-2686. Ko KS, Kim Y-S, Lee MY, Shin SY, Jung DS, Peck KR, Song J-H 2008. Paenibacillus konsidensis sp. nov., isolated from a patient. International journal of Systematic and Evolutionary Microbiology 58(9): 2164-2168. Li B, Ravnskov S, Xie G, Larsen J 2007. Biocontrol of Pythium damping-off in cucumber by arbuscular mycorrhiza-associated bacteria from the genus Paenibacillus. Biocontrol 52(6): 863-875. Liang T-W, Wang S-L 2015. Recent advances in exopolysaccharides from Paenibacillus spp.: production, isolation, structure, and bioactivities. Marine Drugs 13(4): 1847-1863. Mansfeld-Giese K, Larsen J, Bødker L 2002. Bacterial populations associated with mycelium of the arbuscular mycorrhizal fungus Glomus intraradices. FEMS Microbiology Ecology 41(2): 133- 140.

Namjoshi K, Johnson S, Montello P, Pullman GS 2010. Survey of bacterial populations present in US- produced linerboard with high recycle content. Journal of Applied Microbiology 2010 v.108 no.2(no. 2): pp. 416-427. Noskin GA, Suriano T, Collins S, Sesler S, Peterson LR 2001. Paenibacillus macerans pseudobacteremia resulting from contaminated blood culture bottles in a neonatal intensive care unit. American Journal of Infection Control 29(2): 126-129. Oh Y-I, Baek JY, Kim SH, Kang B-J, Youn H-Y 2018. Antimicrobial Susceptibility and Distribution of Multidrug-Resistant Organisms Isolated from Environmental Surfaces and Hands of Healthcare Workers in a Small Animal Hospital. Japanese Journal of Veterinary Research 66(3): 193-202. Oliveira S, Seldin L, Bastos M 1993. Identification of structural nitrogen-fixation (nif) genes in Bacillus polymyxa and Bacillus macerans. World Journal of Microbiology and Biotechnology 9(3): 387- 389. Porter R, McCleskey C, Levine M 1937. The facultative sporulating bacteria producing gas from lactose. Journal of Bacteriology 33(2): 163. Rodriguez-Jerez J, Giaccone V, Colavita G, Parisi E 1994. Bacillus macerans—A new potent histamine producing micro-organism isolated from Italian cheese. Food Microbiology 11(5): 409-415. Rossi F, Dellaglio F 2007. Quality of silages from Italian farms as attested by number and identity of microbial indicators. Journal of Applied Microbiology 2007 v.103 no.5(no. 5): pp. 1707-1715. Sadiq FA, Li Y, Liu T, Flint S, Zhang G, Yuan L, Pei Z, He G 2016. The heat resistance and spoilage potential of aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders. International Journal of Food Microbiology 2016 v.238: pp. 193-201. Sahoo K, Dhal N 2009. Potential microbial diversity in mangrove ecosystems: a review. Savitha K, Raman DS 2012. Isolation, Identification, Resistance profile and Growth kinetics of Chlorpyrifos resistant Bacteria from Agricultural soil of Bangalore. Research in Biotechnology 3(2). Seldin L, Dubnau D 1985. Deoxyribonucleic acid homology among Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and other nitrogen-fixing Bacillus strains. International Journal of Systematic and Evolutionary Microbiology 35(2): 151-154. Teng J, Woo P, Leung K, Lau S, Wong M, Yuen K 2003. Pseudobacteraemia in a patient with neutropenic fever caused by a novel paenibacillus species: Paenibacillus hongkongensis sp. nov. Molecular Pathology 56(1): 29. Tilden EB, Hudson C 1942. Preparation and properties of the amylases produced by Bacillus macerans and Bacillus polymyxa. Journal of Bacteriology 43(4): 527. Vishnoi N, Dixit S, Singh D 2014. Surface binding and intracellular uptake of arsenic in bacteria isolated from arsenic contaminated site. Ecological Engineering 73: 569-578. Wakelin S, Stewart TMA, Walter M 1998. In vitro testing for biological control of Aphanomyces euteiches. Proceedings of the New Zealand Plant Protection Conference. Pp. 90-95. Wakelin SA, Walter M, Jaspers M, Stewart A 2002. Biological control of Aphanomyces euteiches root- rot of pea with spore-forming bacteria. Australasian Plant Pathology 31(4): 401-407. Wenzler E, Kamboj K, Balada-Llasat J-M 2015. Severe sepsis secondary to persistent Lysinibacillus sphaericus, Lysinibacillus fusiformis and Paenibacillus amylolyticus bacteremia. International Journal of Infectious Diseases 35: 93-95. Zhou Y, Gao S, Wei D-Q, Yang L-L, Huang X, He J, Zhang Y-J, Tang S-K, Li W-J 2012. Paenibacillus thermophilus sp. nov., a novel bacterium isolated from a sediment of hot spring in Fujian province, China. Antonie van Leeuwenhoek 102(4): 601-609.

Appendix 1: Revised s26 pathway

Figure 17: Decision pathway for applications under Section 26 for determination as to whether an organism is a new organism

Context

This decision pathway describes the decision-making process for applications under Section 26 for determination as to whether an organism is a new organism.

Introduction

The purpose of this decision pathway is to provide the HSNO decision maker6 with guidance so that all relevant matters in the Hazardous Substances and New Organisms Act (1996) (the Act) and the Hazardous Substances and New Organisms (Organisms Not Genetically Modified) Regulations (1998) (the Regulations) have been addressed. It does not attempt to direct the weighting that the HSNO decision maker may decide to make on individual aspects of an application.

The decision pathway has two parts –

 Flowchart (a logic diagram showing the process prescribed in the HSNO Act and the Methodology to be followed in making a decision), and  Explanatory notes (a discussion of each step of the process).

Of necessity the words in the boxes in the flowchart are brief, and key words are used to summarise the activity required. The explanatory notes provide a description of each of the numbered items in the flowchart, and describe the processes that should be followed.

For proper interpretation of the decision pathway it is important to work through the flowchart in conjunction with the explanatory notes.

6 The HSNO decision maker refers to either the EPA Board or any committee or persons with delegated authority from the Board.

Figure 17 Explanatory Notes

Item 1 Review the content of the application and all relevant information Review the application, staff advice and any relevant information held by other Agencies, and advice from experts.

Item 2 Is further information required? Review the information and determine whether or not there is sufficient information available to make a decision.

Item 3 Seek additional information (Section 52 and Section 58) If the HSNO decision maker considers that further information is required, then this may be sought either from the applicant (if there is an external applicant) or from other sources. If the HSNO decision maker considers that the information may not be complete but that no additional information is currently available, then the HSNO decision maker may proceed to make a determination. If the application is not approved on the basis of lack of information (or if the organism is considered new) and further information becomes available at a later time, then the HSNO decision maker may choose to revisit this determination.

Item 4 Is it an organism (i.e. fits the “organism” definition in Section 2)? An organism

(a) does not include a human being: (ab) includes a human cell: (b) includes a micro-organism: (c) includes a genetic structure, other than a human cell, that is capable of replicating itself, whether that structure comprises all or only part of an entity, and whether it comprises all or only part of the total genetic structure of an entity: (d) includes an entity (other than a human being) declared to be an organism for the purposes of the Biosecurity Act 1993: (e) includes a reproductive cell or developmental stage of an organism

If yes, go to item 5. If no, as this is not an organism, it is not regulated under the new organism provisions of the HSNO Act.

Item 5 Is the determination about a potential GMO (Section 2A(1)(d))? If the determination relates to whether an organism is a potential GMO, go to pathway B. If the organism is not a GMO, go to item 6.

Item 6 Does the organism belong to a species that was known to be present in NZ immediately before 29 July 1998 (Section 2A(1)(a))? Determine on the basis of the available information whether on balance of probabilities the organism is known to belong to a species that was present in New Zealand immediately prior to 29 July 1998. For the purposes of making a Section 26 determination an organism is considered to be present in New Zealand if it can be established that the organism was in New Zealand: (a) immediately before 29 July 1998; and (b) not in contravention of the Animals Act 1967 or the Plants Act 1970 (excluding rabbit haemorrhagic disease virus, or rabbit calicivirus). If yes, go to item 7 to test the organism against the next criterion. If no, go to item 12.

Item 7 Is the organism prescribed as a risk species and was not present in New Zealand at the time of promulgation of the relevant regulation (Section 2A(1)(b))? Determine whether the organism belongs to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been prescribed as a risk species by regulation established under Section 140(1)(h) of the Act. If the organism is prescribed as a risk species, determine whether it was present in New Zealand when it was prescribed. The organism is a new organism if it was not present in New Zealand at the time of the promulgation of the relevant regulation. Note: at this point it may become apparent that the organism is an unwanted organism under the Biosecurity Act. If this is the case, then MPI and DOC may be advised (they may already have been consulted under items 1, 2 and 3). If yes, go 12. If no, go to item 8 to test the organism against the next criterion.

Item 8 Has a containment approval been given for the organism under the Act (Section 2A(1)(c))? For the purposes of making a Section 26 determination, this will also include the following organisms which are “deemed” to be new organisms with containment approvals under the HSNO Act: (a) animals lawfully imported under the Animals Act 1967 before 29 July 1998 pursuant to Section 254 of the HSNO Act; (b) animals lawfully present in New Zealand in a place that was registered as a zoo or circus under the Zoological Garden Regulations 1977 pursuant to Section 255 of the HSNO Act (except where other organisms of the same taxonomic classification were lawfully present outside of a zoo or circus –see section 2A(2)(c)); (c) hamsters lawfully imported under the Hamster Importation and Control Regulations 1972 pursuant to Section 256 of the HSNO Act; or (d) plants lawfully imported under the Plants Act 1970 before 29 July 1998 pursuant to Section 258 of the HSNO Act. If yes, go to item 12. If no, go to item 9 to test the organism against the next criterion.

Item 9 Has a conditional release approval been given for the organism (Section 2A(1)(ca))?

If yes, go to item 12. If no, go to item 10 to test the organism against the next criterion.

Item 10 Has a qualifying organism with controls approval been given for the organism (Section 2A(1)(cb))? A “qualifying organism” is an organism that is or is contained in a “qualifying medicine” or “qualifying veterinary medicine”. These terms are defined in Section 2 of the HSNO Act. If yes, go to item 12. If no, go to item 11 to test the organism against the next criterion.

Item 11 Is the organism known to have been previously eradicated (Section 2A(1)(e))? Determine whether the organism belongs to a species, subspecies, infrasubspecies, variety, strain, or cultivar that is known to have been previously eradicated. Eradication does not include extinction by natural means but is considered to be the result of a deliberate act. If yes, go to item 12. If no, then the organism is not a new organism.

Item 12 Has HSNO release approval without controls been given for an organism of the same taxonomic classification under Sections 35, 38 or 38I of the Act or has an organism of the same taxonomic classification been prescribed as a not new organism (Section 2A(2)(a))?

If a release approval has been given for an organism of the same taxonomic classification under Section 35 or 38 of the Act then the organism is not a new organism. If a release approval has been given for an organism of the same taxonomic classification under Section 38I of the Act without controls then the organism is not a new organism, however, if this approval has been given with controls then it is a new organism. If an organism of the same taxonomic classification has been prescribed by regulations as not a new organism7 then it is not a new organism. If yes, the organism is not a new organism.If no, the organism is a new organism.

7 http://www.legislation.govt.nz/regulation/public/2009/0143/latest/whole.html#DLM2011201