APP203514 Soil Fungus Import and Release Submissions

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APP203514 Soil fungus import and release Submissions

29 November 2018

Under section 34 of the Hazardous Substances and New Organisms Act 1996 Volume 1 of 1

To import and release the arbuscular mycorrhizal fungus Glomus iranicum var. tenuihypharum in New Zealand

Submission Number Submitter Submitter Organisation

SUBMISSION 127379 Clinton Care

SUBMISSION 127403 Ian Dickie

SUBMISSION 127407 Gerry Coates Te Runanga o Ngai Tahu

SUBMISSION 127408 Peter Buchanan Landcare Research NZ Ltd

SUBMISSION 127409 Rod Hitchmough Dept of Conservation

SUBMISSION 127410 Cliff Mason

1 SUBMISSION 127379

From: Account Update [mailto:[email protected]] Sent: Friday, 19 October 2018 10:28 PM To: submissions Subject: Re: APP203514 Application open for public submissions - Soil fungus

Hi Diane, Regarding importation and release the arbuscular mycorrhizal fungus Glomus iranicum var. tenuihypharum in New Zealand...

I think ... be careful about exotic soil fungus as it may kill native worms and earthworms or other fungus in soil.

One of our native worm were imported to Britain and these worms ate many earthworms in British soil.

Try testing the exotic soil fungus in a large pot of soil, with earthworms.

Yours Sincerely

Clinton Care.

1 SUBMISSION 127403

SUBMISSION FORM For Hazardous Substance and New Organism Applications

Once you have completed this form Send by post to: Environmental Protection Authority, Private Bag 63002, Wellington 6140 OR email to: [email protected]

Once your submission has been received the submission becomes a public document and may be made publicly available to anyone who requests it. You may request that your contact details be kept confidential, but your name, organisation and your submission itself will become a public document.

Submission on application APP203514 number: Name of submitter or contact for Prof. Ian A. Dickie joint submission: Organisation name (if on behalf of an organisation): Postal address: School of Biological Sciences, University of Canterbury Private Bag 4800 Christchurch 8140

Telephone number: 033692268

Email: [email protected]

I wish to keep my contact details confidential

The EPA will deal with any personal information you supply in your submission in accordance with the Privacy Act 1993. We will use your contact details for the purposes of processing the application that it relates to (or in exceptional situations for other reasons permitted under the Privacy Act 1993). Where your submission is made publicly available, your contact details will be removed only if you have indicated this as your preference in the tick box above. We may also use your contact details for the purpose of requesting your participation in customer surveys.

The EPA is likely to post your submission on its website at www.epa.govt.nz. We also may make your submission available in response to a request under the Official Information Act 1982.

www.epa.govt.nz 2

Submission Form

I support the application

I oppose the application

I neither support or oppose the application

The reasons for making my submission are1: (further information can be appended to your submission, see footnote). In my professional opinion as a researcher with 18 years of experience in mycorrhizal fungi, and over 10 years of experience working with invasive species in New Zealand, the proposal has over-stated the benefits of this introduction and under-stated the potential for harm. Please see attached document for my complete assessment of the proposal.

All submissions are taken into account by the decision makers. In addition, please indicate whether or not you also wish to speak at a hearing if one is held. I wish to be heard in support of my submission (this means that you can speak at the hearing) I do not wish to be heard in support of my submission (this means that you cannot speak at the hearing) If neither box is ticked, it will be assumed you do not wish to appear at a hearing.

I wish for the EPA to make the following decision: The application should be declined. Please see the attached document for further information.

1 Further information can be appended to your submission, if you are sending this submission electronically and attaching a file we accept the following formats – Microsoft Word, Text, PDF, ZIP, JPEG and JPG. The file must be not more than 8Mb.

July 2016 EPA0190 Hazardous Substances and New Organisms Environmental Protection Authority Private Bag 63002 Wellington 6140 [email protected]

Re: APP203514 Application open for public submissions - Soil fungus

I am writing this submission in opposition to APP203514, a proposal to introduce Glomus iranicum var. tenuihypharum (syn. Dominikia iranica), on the basis of my professional expertise as Professor of Microbial Ecology at the University of Canterbury and a project leader at the Bio-Protection Research Centre. The views I present are my own, informed by my research experience, specifically in the area of mycorrhizal ecology, including publications on invasive mycorrhizal fungi and the risks associated with introducing fungal symbionts. The proposal is to introduce Glomus iranicum var. tenuihypharum, which forms arbuscular mycorrhizal associations with plants. The fungus was isolated in Spain, but the same species has been found in multiple countries. A mixture of this variety of fungus with clay has been patented (US2017/0188587). It should also be noted that the species name provided in the application is no longer current, as Glomus iranicum is now understood to be Dominikia iranica (Blaszkowski et al. 2015). I make the following notes about this proposed introduction:

1. The benefits of this introduction are overstated and risks are considerable Arbuscular mycorrhizal fungi are naturally present in soils, with multiple species typically co- occurring. As a group, these fungi are highly beneficial to most plants and are an important component of the soil ecosystem, including enhancing soil structure. There is no question that arbuscular mycorrhizal symbioses are beneficial under most natural circumstances. Nonetheless, arbuscular mycorrhizal fungi are already present in the majority of soils at levels sufficient to meet plant needs for mycorrhizal associations. In the relatively uncommon situation where mycorrhizal inoculum potential in soils is low (e.g., following a Brassica rotation or agricultural conversion from pine plantation), there are already available products. Mycorrhizal inoculum products are sold worldwide, including in New Zealand. These include Mycormax (https://www.rd2.co.nz/product/mycormax-2/), and Myco-Gro (http://flrc.massey.ac.nz/workshops/15/Manuscripts/Paper_Monk_2015.pdf). Other, already-available, products claim to enhance indigenous arbuscular mycorrhizal inoculation potential of agricultural soils (http://biostart.co.nz/products/soil-microbial- activators/mycorrcin/). As such, the introduction of this new species would not provide a service that is currently lacking in New Zealand. There is a long history of many mycorrhizal inoculum products failing to show much benefit to plants under field conditions. This often reflects problems with storage and shipping of inoculum, and/or inappropriate application. Further, the presence of established fungal communities in soils is a strong barrier to successful introduction and maintenance of introduced strains. Finally, in many agricultural settings, the overall benefits of mycorrhizal fungi to plants may be small, with a recent comprehensive review finding little evidence that farmers need be concerned about mycorrhizas under typical farm conditions (Ryan and Graham 2018). This largely reflects the high carbon cost of supporting mycorrhizal fungi, and relatively minor benefits under high P conditions. There are also reports that arbuscular mycorrhizal fungi can become parasitic on agricultural crops (Hendrix et al. 1992), particularly under high nutrient situations (Buwalda and Goh 1982; Peng et al. 1993; Ryan and Graham 2002; Lekberg and Koide 2005; Grace et al. 2009). At least one recent study has found that the abundance of Glomus iranicum is negatively correlated with wheat production in organic agriculture, which the authors attribute to a high carbon demand of the fungus from the host (Dai et al. 2014). As such, it cannot be assumed that introducing Glomus iranicum will enhance agricultural productivity, and, indeed, there is some evidence that it could put agricultural productivity at risk.

2. The fungus will spread widely In the submission to introduce the species, it is claimed that “its spread is confined to the application zone”. This is not supported by scientific evidence, and I suggest that Glomus iranicum is almost certain to spread beyond the site of introduction. Arbuscular mycorrhizal fungi produce spores and hyphal fragments that are dispersed by soil and root movements, rodents (Mangan and Adler 1999), birds (Nielsen et al. 2016; Correia et al. 2018), and wind (Warner et al. 1987; Egan et al. 2014; de Leon et al. 2016). The suggestion in the application that fungi only spread by root-to-root contact is provably false on the basis of scientific literature. Any applied spores are therefore likely to spread widely into adjacent ecosystems. The plant-host specificity of arbuscular mycorrhizal fungi is low, hence once they disperse it is highly likely that the fungus will associate with a broad range of plants, including in native ecosystems.

3. There will likely be impacts on native fungal communities Fungi, like all organisms, compete for resources. If successfully introduced Glomus iranicum will inevitably compete with other fungi for space on plant roots (Wilson 1984; Hepper et al. 1988) and potentially for soil resources. Thus the introduction of Glomus iranicum is likely to reduce populations of native arbuscular mycorrhizal fungi, many of which species are still unknown to science. High populations of Glomus iranicum may also support parasites and pathogens that impact directly on native arbuscular mycorrhizal fungi (pathogen spill-over and spill-back).

4. The introduction may compromise native fungal genetics In addition to ecological interactions (competition, pathogen spill-over and spill-back), introduced Glomus iranicum may have the potential to hybridize with native fungi. The reproductive life-cycle of Glomus remains unknown. While arbuscular mycorrhizal fungi have a high production of asexual spores, there is some evidence that sexual reproduction may also take place. Further, arbuscular mycorrhizal fungi such as Glomus are able to show anastomosis, where hyphae of more than one individual fuse and share cellular contents (Parniske 2008). It has been (controversially) suggested that this can lead to fungal spores containing nuclei from multiple species. Given how little is known about the reproductive cycle of Glomus, these potential impacts remain difficult to quantify, but should be considered in detail before permitting any new species introductions.

5. The introduction is likely to change plant communities Arbuscular mycorrhizal fungi show a degree of plant-host preference, with different species of fungi occurring on different plant hosts (Martinez‐Garcia et al. 2015). Fungi may also differ in the degree to which they benefit one plant host over another. It is widely recognized that arbuscular mycorrhizal fungi can cause growth depressions in some plant species and that benefits provided to plants are highly unequal across plant species (Klironomos 2003; Schwartz et al. 2006). The spread of Glomus iranicum is therefore likely to cause changes in the competitive hierarchies of plants, allowing some plant species to become more dominant at the expense of others. This is particularly a risk in terms of facilitating non-native plant invasions. The introduction of non-native fungi is considered to be responsible for facilitating the invasion of wilding pines (Pinus contorta) and other invasive plants in New Zealand (Dickie et al. 2010; Dickie et al. 2017), a process that has also been observed in international research (Hynson et al. 2013; Hayward et al. 2015).

6. The introduction will be irreversible Introducing fungi would likely be an irreversible decision. There are no known examples where an introduced fungus has been successfully eliminated once established (Dickie et al. 2016). Hence, should Glomus iranicum be introduced, it will likely be impossible to remove the species at a later date. The suggestion from the proposal that removing plant roots and spraying fungicide would eradicate Glomus iranicum is based on the assumptions that (a) there will be no spread, (b) all roots can be physically removed from soil, and (c) that fungicide is effective against Glomus. None of these assumptions have been shown to be true. In summary, the benefits of allowing this introduction are likely to be minimal, the risk of spread and impacts on native fungal and plant communities are high, and any introduction will likely be an irreversible decision. The application to introduce the organism under-states the potential for spread, fails to consider potential impacts on native plant and fungal communities, and incorrectly states that removal would be possible. From my professional viewpoint, the risks are vastly greater than any potential benefits from this proposed introduction. On that basis, and on the basis of a fairly extensive scientific literature on the topic of invasive mycorrhizal fungi (Schwartz et al. 2006; Nunez and Dickie 2014; Nunez et al. 2015; Dickie et al. 2016; Dickie et al. 2017), I believe the application should be declined.

Signed,

Ian Dickie, Professor of Microbial Ecology, School of Biological Sciences, University of Canterbury

References

Blaszkowski J, Chwat G, Góralska A, Ryszka P, Kovács GM 2015. Two new genera, Dominikia and Kamienskia, and D. disticha sp. nov. in Glomeromycota. Nova Hedwigia 100: 225–238. Buwalda JG, Goh KM 1982. Host-fungus competition for carbon as a cause of growth depressions in vesicular-arbuscular mycorrhizal ryegrass. Soil Biology and Biochemistry 14: 103–106. Correia M, Heleno R, da Silva LP, Costa JM, Rodríguez‐Echeverría S 2018. First evidence for the joint dispersal of mycorrhizal fungi and plant diaspores by birds. New Phytologist Dai M, Hamel C, Bainard LD, Arnaud MS, Grant CA, Lupwayi NZ, Malhi SS, Lemke R 2014. Negative and positive contributions of arbuscular mycorrhizal fungal taxa to wheat production and nutrient uptake efficiency in organic and conventional systems in the Canadian prairie. Soil Biology and Biochemistry 74: 156–166. de Leon DG, Moora M, Öpik M, Jairus T, Neuenkamp L, Vasar M, Bueno CG, Gerz M, Davison J et al. 2016. Dispersal of arbuscular mycorrhizal fungi and plants during succession. Acta oecologica 77: 128–135. Dickie IA, Bolstridge N, Cooper JA, Peltzer DA 2010. Co-invasion by Pinus and its mycorrhizal fungi. New Phytologist 187: 475–484. Dickie IA, Bufford JL, Cobb RC, Desprez‐Loustau M, Grelet G, Hulme PE, Klironomos J, Makiola A, Nuñez MA et al. 2017. The emerging science of linked plant–fungal invasions. New Phytologist 215: 1314–1332. Dickie IA, Nuñez MA, Pringle A, Lebel T, Tourtellot SG, Johnston PR 2016. Towards management of invasive ectomycorrhizal fungi. Biological Invasions 18: 3383–3395. Egan C, Li D-W, Klironomos J 2014. Detection of arbuscular mycorrhizal fungal spores in the air across different biomes and ecoregions. Fungal Ecology 12: 26–31. Grace EJ, Cotsaftis O, Tester M, Smith FA, Smith SE 2009. Arbuscular mycorrhizal inhibition of growth in barley cannot be attributed to extent of colonization, fungal phosphorus uptake or effects on expression of plant phosphate transporter genes. New Phytologist 181: 938–949. Hayward J, Horton TR, Pauchard A, NUNez MARTINA 2015. A single ectomycorrhizal fungal species can enable a Pinus invasion. Ecology 96: 1438–1444. Hendrix JW, Jones KJ, Nesmith WC 1992. Control of pathogenic mycorrhizal fungi in maintenance of soil productivity by crop rotation. Journal of Production Agriculture 5: 383– 386. Hepper CM, Azcon‐Aguilar C, Rosendahl S, Sen R 1988. Competition between three species of Glomus used as spatially separated introduced and indigenous mycorrhizal inocula for leek (Allium porrum L.). New Phytologist 110: 207–215. Hynson NA, Merckx VSFT, Perry BA, Treseder KK 2013. Identities and distributions of the co-invading ectomycorrhizal fungal symbionts of exotic pines in the Hawaiian Islands. Biol Invasions Klironomos JN 2003. Variation in plant response to native and exotic arbuscular mycorrhizal fungi. Ecology 84: 2292–2301. Lekberg Y, Koide RT 2005. Is plant performance limited by arbundance of arbuscular mycorrhizal fungi? A meta-analysis of studies published between 1988 and 2003. New Phytologist 168: 189–204. Mangan SA, Adler GH 1999. Consumption of arbuscular mycorrhizal fungi by spiny rats (Proechimys semispinosus) in eight isolated populations. Journal of Tropical Ecology 15: 779–790. Martinez‐Garcia LB, Richardson SJ, Tylianakis JM, Peltzer DA, Dickie IA 2015. Host identity is a dominant driver of mycorrhizal fungal community composition during ecosystem development. New Phytologist 205: 1565–1576. Nielsen KB, Kjøller R, Bruun HH, Schnoor TK, Rosendahl S 2016. Colonization of new land by arbuscular mycorrhizal fungi. Fungal Ecology 20: 22–29. Nunez MA, Dickie IA 2014. Invasive belowground mutualists of woody plants. Biological Invasions 16: 645–661. Nunez MA, Dimarco RD, Dickie IA, Pauchard A 2015. ¿ Qué puede salir mal?: Los riesgos de introducir microorganismos del suelo de la Antártida en América del Sur. Bosque (Valdivia) 36: 343–346. Parniske M 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Reviews Microbiology 6: 763. Peng S, Eissenstat DM, Graham JH, Williams K, Hodge NC 1993. Growth depression in mycorrhizal citrus at high-phosphorous supply. Plant Physiology 101: 1063–1071. Ryan MH, Graham JH 2002. Is there a role for arbuscular mycorrhizal fungi in production agriculture? Plant and Soil 244: 263–271. Ryan MH, Graham JH 2018. Little evidence that farmers should consider abundance or diversity of arbuscular mycorrhizal fungi when managing crops. New Phytologist Schwartz MW, Hoeksema JD, Gehring CA, Johnson NC, Klironomos JN, Abbott LK, Pringle A 2006. The promise and the potential consequences of the global transport of mycorrhizal fungal inoculum. Ecology Letters 9: 501–515. Warner NJ, Allen MF, MacMahon JA 1987. Dispersal agents of vesicular-arbuscular mycorrhizal fungi in a disturbed arid ecosystem. Mycologia 721–730. Wilson JM 1984. Competition for infection between vesicular‐arbuscular mycorrhizal fungi. New Phytologist 97: 427–435.

SUBMISSION 127407

He tono nā

ki te ENVIRONMENTAL PROTECTION AUTHORITY

e pā ana ki te SUBMISSION ON APP203514 – For approval to release the arbuscular mycorrhizal soil fungus Glomus iranicum var. tenuihypharum.

26 November 2018

Author – Gerry Te Kapa Coates Ngāi Tahu HSNO Komiti

Sponsor – Kara Edwards General Manager – Te Ao Tūroa | Te Kaihautū o Te Ihu Waka I Te Rūnanga o Ngāi Tahu [email protected] I Phone 03 366 4344 I PO Box 13-046 I Christchurch

Oppose – conditional request to be heard

Contents

1. EXECUTIVE SUMMARY 2. ABOUT TE RŪNANGA O NGĀI TAHU 3. TE RŪNANGA STATEMENTS OF POSITION 4. RECOMMENDATIONS ON APP203514

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1. EXECUTIVE SUMMARY

Over the years, Te Rūnanga o Ngāi Tahu (TRoNT) has advocated for restraint in approving new organisms, particularly those resulting from genetic modification or those that can form self- sustaining populations. We are usually generally supportive of any proposal which may decrease the burden of pesticide residues or their breakdown products on the biotic and abiotic environments. In this case the applicant has been quite open that they are applying for the approval to release a new micro-organism, an arbuscular mycorrhizal fungus (AMF) (Glomus iranicum tenuihypharum) in New Zealand “to start to commercialize our products, based on this AMF, in the New Zealand’s market.” They say this is “related to the great market opportunity that New Zealand” linking it to guaranteeing a sustainable future. The applicant has provided no economic estimates of the potential benefits to New Zealand or commented on any possible environmental risks and costs.

Pre-application attempts at consultation with Māori relating to this biocontrol agent were carried out with Māori, including Ngāi Tahu, questioning whether competition with any native Glomus spp. might occur and whether there could be any deleterious effect on native fauna and flora. We believe these risks should have been assessed and where possible been adequately quantified and compared with the benefits.

The AMF is stated not to be a genetically modified organism. It can be used for soil stabilization or as a plant improver through soil maximization and protection with a reduced need for fertilisation. It can be applied through irrigation systems, or in a powder form designed to be used as a seed coating or in a microgranular form designed to be applied through an applicator directly in the planting furrow. The AMF cannot develop outside a host plant and dissemination of the arbuscular mycorrhizal fungus is low since they are lacking aerial spores and only propagate by contact of the spores, the vesicles or the mycelium with the roots of a new host plant. However we wonder if it can also be disseminated – like Kauri dieback disease in NZ – by human and animal traffic.

Our submission generally opposes the Application on the grounds that provided it is primarily for the economic benefit of the applicant’s company as well as for the soil health benefits. No need has been demonstrated and no commentary on the possibility of using NZ Native species of such fungi was provided.

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2. TE RŪNANGA O NGĀI TAHU

2.1. This response is made on behalf of Te Rūnanga o Ngāi Tahu (Te Rūnanga). Te Rūnanga is statutorily recognised as the representative tribal body of Ngāi Tahu Whānui and was established as a body corporate on 24th April 1996 under section 6 of Te Rūnanga o Ngāi Tahu Act 1996 (the Act). We note the following relevant provisions of our constitutional documents:

a) Section 3 of the Act States: This Act binds the Crown and every person (including any body politic or corporate) whose rights are affected by any provisions of this Act.

b) Section 15(1) of the Act states: Te Rūnanga o Ngāi Tahu shall be recognised for all purposes as the representative of Ngāi Tahu Whānui.

c) The Charter of Te Rūnanga o Ngāi Tahu (1993, as amended) constitutes Te Rūnanga as the kaitiaki of the tribal interest.

2.2. Ngāi Tahu is the third largest Māori iwi in Aotearoa with a membership of almost 60,000 who whakapapa to an ancestor in the 1848 census of tūpuna. Its takiwā (area of influence) extends from Kaikōura in the north, to Rakiura (Stewart Island) in the south, including the West Coast, Te Tai Poutini. This comprises over 90% of the South Island or over 40% of the NZ land mass. Te Rūnanga o Ngāi Tahu is statutorily recognised as the representative tribal body of Ngāi Tahu Whānui under section 6 of Te Rūnanga o Ngāi Tahu Act 1996. This means it exercises kaitiakitanga over this takiwā.

2.3. Te Rūnanga o Ngāi Tahu constitutes 18 Rūnanga representing geographical areas, generally based around traditional settlements.

2.4. Ngāi Tahu Values which dictate its approach to all issues are as follows:

· Whanaungatanga (family) Respect, foster and maintain important relationships within the organisation, within the iwi and within the community.

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· Manaakitanga (looking after our people) Respect each other, iwi members and all others in accordance with our tikanga (customs). · Tohungatanga (expertise) Pursue knowledge and ideas that will strengthen and grow Ngāi Tahu and our community. · Kaitiakitanga (stewardship) Work actively to protect the people, environment, knowledge, culture, language and resources important to Ngāi Tahu for future generations. · Tikanga (appropriate action) Strive to ensure that Ngāi Tahu tikanga is actioned and acknowledged in all of our outcomes. · Rangatiratanga (leadership) Strive to maintain a high degree of personal integrity and ethical behaviour in all actions and decisions we undertake.

2.5. Te Rūnanga respectfully requests that this response is accorded the status and weight due to the mana whenua status of the tribal collective, Ngāi Tahu Whānui.

3. TE RŪNANGA STATEMENTS OF POSITION ON APP203514

3.1. General position on new organisms Over the years, Te Rūnanga o Ngāi Tahu (TRoNT) has advocated for restraint in approving new organisms, particularly those resulting from genetic modification or those that can form self-sustaining populations. We are usually generally supportive of any proposal which may decrease the burden of pesticide residues or their breakdown products on the biotic and abiotic environments. In this case the applicant has been open that they “are applying for the approval to release a new micro-organism, an arbuscular mycorrhizal fungus (AMF) (Glomus iranicum tenuihypharum) in New Zealand to start to commercialize our products, based on this AMF, in the New Zealand’s market. The fact that we want to release our products in New Zealand are related to the great market opportunity that New Zealand represents and to the improve (sic) of agricultural systems to guarantee a sustainable future.”

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In other words this fungus that was originally endemic in Spain is being introduced primarily for business reasons, and our task is to assess whether:

· There is any risk from introducing such a new organism into New Zealand. · Whether there are any benefits to be gained from such an introduction. · Whether such beneficial outcomes can be gained by using similar NZ sourced fungi of the same genera.

3.2. Māori Reference Group A Māori Reference Group (MRG) was formed in 2014 specifically to consider biocontrol applications. The aims of biological control – which were fully supported by the MRG and by Ngāi Tahu – is to reduce risk and reverse harm from damaging organisms, as one of the tools for pest management. The MRG developed some core principles which have also been referred to in the Application. They also apply to this type of application where a new organism is to be introduced ostensibly to improve the fertility of soil.

The species Glomus iranicum has not been detected in New Zealand so far, but this does not mean it is exotic since closely phylogenetic species have been found. This may be related to the lack of studies carried out in New Zealand or to a misidentification of the species. The applicant says that “non-target effects of biofertilizers are often small and transient, the soil system has been found to be resilient to perturbations caused by introduction of exogenous AMF.”

Our summary of the principles for biocontrol – which also apply to new organisms – were as follows:

3.2.1. MRG PRINCIPLES:

Kaitiakitanga

There is a well-recognised kaitiakitanga responsibility for Māori to manage the natural resources within and beyond their hapū and iwi boundaries for the benefit of future generations.

Manaakitanga

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Biocontrol agents pose the potential to both positively impact by aiding in the restoration of balance, and negatively impact by disturbing it further. Good decision-making is thus crucial. If appropriate, regional councils and the Department of Conservation should work with iwi and hapū in their areas on pest management strategies that include monitoring impacts in terms of manaakitanga.

3.2.2. Broad biophysical considerations

Māori will be concerned to know the anticipated and unanticipated potential impact of the introduction of biocontrol agents across the breadth of trophic and ecosystem levels.

Specific impacts on culturally valued species

The reference group recognised that standard host range testing and taxonomical analysis provides data that gives some assurance that any direct adverse effect from the non- target feeding and hybridisation of native species is likely to be minimal.

However the research methodology and results do little to address indirect impacts to culturally valued species.

It is thus important to continue to monitor potential effects (adverse and otherwise) of new introduced supposedly beneficial species on closely related native species.

Selected MRG Recommendations

i) Applicants should provide comment on or model the potentially broader trophic impacts of introducing a biological control agent. This has been done in the Application to some degree. ii) Applicants should provide information about the potential beneficial role a new introduced species may have for local populations of native species. This has been done in this Application in general terms. iii) Applicants should ensure recognition and assessment of impacts (both positive and negative) against appropriate national and regional Treaty principles and provisions.

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3.3. Ngāi Tahu Comments on the Application

General aspects

3.3.1. Biological activities in soil can play a vital part in nutrient cycling and availability to plants and in soil structure, contributing to soil health. Sustainability in land-use requires minimising soil degradation and adapting soil management practices to conserve and augment soil resources.

3.3.2. The Applicant says that the Glomus species is “found all over the world in soil or in edible roots (carrots for example and no case of toxicity, pathogenicity or mention of a glomus toxin can be found in the literature and no signs of toxicity have been seen in research or technical personnel working on the endomycorrhiza during personnel monitoring.” Glomus iranicum var. tenuihypharum is stated not to be a genetically modified organism, the particular strain was isolated from a soil with high salinity in Spain. High salinity in soils is usually a significant drawback for agriculture significantly reducing foliar growth.

3.3.3. Also as an obligatory symbiont, this species, like other Glomalean fungi, cannot develop outside a host plant and dissemination of the arbuscular mycorrhizal fungus is low since they are lacking aerial spores and only propagate by contact of the spores, the vesicles or the mycelium with the roots of a new host plant. Therefore, its dissemination in the environment is limited to the application zone. We wonder however if it can also be disseminated like Kauri dieback disease in NZ by human and animal traffic. Reports apparently show that organic and mineral fertilisation, tilling, monoculture and other agricultural practices all negatively affect AMF abundance and general biodiversity.

3.3.4. AMF can be used for soil stabilization or as a plant improver through:

· Soil maximization and protection with a reduced need for fertilisation.

· Increasing tolerance to drought and/or salinity.

3.3.5. The applicant has developed several formulations: a powder form applied through irrigation systems; a powder form designed to be used as a seed coating; and. a microgranular form designed to be applied through an applicator directly in the furrow.

3.3.6. Where soil conditions have been degraded by the intensive use of chemicals and bad agricultural practices they can be rejuvenated by inoculating the soils with microorganisms, such as G. i. tenuihypharum. The applicant says that such usage is certified in several countries for the use in organic agriculture.

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3.3.7. The applicant says that G. i. tenuihypharum is unable to form self-sustaining populations as an obligate symbiont, depending on association with plant roots to complete their life cycle. However its eradication can be simply achieved by the complete removal of all the roots from the soil, and/or application of soil sanitisation treatments such as fumigation or fungicides.

Māori engagement

3.3.8. The applicant says that Iwi and Māori communities were contacted, as advised by their EPA consultant. This included emails presented to both the Ngāi Tahu and the Ngāpuhi HSNO Komitis and also Ngāti Huarere ki Whangapoua representatives for consultation, They say they received two replies. “Ngāi Tahu showed a deep interest in the introduction of our Glomus spp. into the New Zealand environment. The only concerns were about the competition with native Glomus spp. and any deleterious effect on native fauna and flora.” They say an explanatory answer about these concerns was presented, although we have no record of this, and they say no answer was obtained thus concluding the consultation.

3.3.9. A similar answer was apparently obtained from the Te Herenga network although this correspondence was not included in the application.

Risks, costs and benefits

3.3.10. The applicant says “The effects of mycorrhizal associations on agricultural and horticultural systems are almost all potentially beneficial.” The non-beneficial elements were not identified. “Non-nutritional” effects in stabilizing soil aggregates, and in preventing erosion, and alleviating negative effects induced by salinity” are mentioned however.

Monitoring the release

3.3.11. If the release is approved then a monitoring plan to check the AMF’s efficacy and confirm it does not spread should be carried out by the applicant or another contracted party.

4. CONCLUSIONS AND RECOMMENDATIONS

4.1. Te Rūnanga o Ngāi Tahu holds long-standing concerns over the introduction of new organisms unless the risks are properly assessed, as opposed to any perceived benefits. Generally we have ended up supporting such applications particularly if they are for

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biocontrol. However in this case where the application is primarily for the economic advantage of the applicant and no compelling positive case or need for the introduction is made at best we can remain neutral or oppose the application.

4.2. Because this fungus is being introduced primarily for business reasons, as well as some unquantified soil benefits, our task is to assess whether:

4.3. We believe the question of risk has not been adequately addressed in the application. It is not sufficient to assert that “The effects of mycorrhizal associations on agricultural and horticultural systems are almost all potentially beneficial” without identifying what the non-beneficial effects are. Furthermore the benefits to NZ need to be quantified in some way, as well as attributed to a new or existing need.

4.4. The question we raised in pre-application consultations regarding whether “competition with native Glomus spp. and any deleterious effects on native fauna and flora” were likely to arise, have not been satisfactorily answered.

4.5. In this case Ngāi Tahu has decided to oppose the Application on the grounds that an economic case has not been made for the introduction of a new exotic organism for soil stabilization or as a plant improver. In our view the risks that have been alluded to such as effects being “almost all potentially beneficial” have not been stated or quantified.

4.6. We have therefore decided to Oppose this application and to reserve our right to be heard and to make any concerns we have in person, if a hearing is convened for other submitters.

Page | 10

5. RECOMMENDATION

5.1. Te Rūnanga o Ngāi Tahu Opposes the Applicant’s proposal to release the arbuscular mycorrhizal soil fungus Glomus iranicum var. tenuihypharum.

Gerry Te Kapa Coates MNZM Member, Ngā Tahu HSNO Committee

Email: [email protected] Mobile: 021 355099

Page | 11

SUBMISSION 127408

SUBMISSION FORM For Hazardous Substance and New Organism Applications

Once you have completed this form Send by post to: Environmental Protection Authority, Private Bag 63002, Wellington 6140 OR email to: [email protected]

Submission on application APP203514 number: Name of submitter or contact for Peter Buchanan joint submission: Organisation name Manaaki Whenua - Landcare Research (if on behalf of an organisation): Postal address: Private Bag 92170, Auckland 1142

Telephone number:

Email:

I wish to keep my contact details confidential

The EPA is likely to post your submission on its website at www.epa.govt.nz. We also may make your submission available in response to a request under the Official Information Act 1982.

www.epa.govt.nz 2

Submission Form

I support the application

I oppose the application

I neither support or oppose the application

The reasons for making my submission are1: (further information can be appended to your submission, see footnote). The application relates to an organism that is new to New Zealand, but consideration is also needed of the patented product(s) that contain this organism The host range of G. iranicum var. tenuihypharum is reported to be large, but is unknown. The variety is reported to be cosmopolitan, but evidence for this is not provided. The patented product contains various biocides (chemicals and live organisms) that will need to be assessed by EPA The potential competition and other effects of the new organism on native Glomus and other mycorrhizal fungi cannot be assumed or assessed. Claims of easy eradication of the new organism if required appear to be unrealistic. The carrier materials comprise about 96-99% of the product but are unlikely to be sterile? Is there a risk of importation of unknowns as contaminants? I note that while the accepted name for Glomus iranicum is now Dominikia iranica, the variety in question, var. tenuihypharum, has not (yet) been recombined in Dominikia. Please see additional information and commentary appended to this submission.

I wish for the EPA to make the following decision: I think this organism, and the patented products that contain it, pose unacceptable biosecurity and biodiversity risk to NZ, I request that EPA declines this application.

July 2016 EPA0190 Additional comment relating to my Submission on EPA Application no. APP203514

The following is additional information and commentary to be appended to my submission.

I first refer to selected quoted text from the EPA Application by pagination or by sections (where pagination is disrupted, eg, reverting to “10” at p. 16 …), followed by Comments.

Also attached is detail and questions relating to the composition of the patented product containing Glomus iranicum var. tenuihypharum, and to details about the marketed product MycoUp.

Extracted text from APP203514: p. 7. “Being an obligatory symbiont, this species, like other Glomalean fungi, cannot develop outside a host plant.” …. “its dissemination in the environment is limited to the application zone.”

Comment: Spores of this fungus exist external to the host and hence can be moved in soil. Evidence that such spores can be viable is implicit in the development of Symborg’s products. Host range is unknown, and in the NZ context unknowable. The “application zone” could be of small to large scale depending on how the farmer, orchardist, gardener, nurseryman, or perhaps restoration planter chooses to apply the product. In the Safety Data Sheet relating to Symborg’s MycoUp product that contains this organism, I note a warning to avoid spread and runoff into waterways (see below).

p. 10-11. “According to this phylogeny Glomus indicum and Glomus achrum are the closest relatives. At the NCBI server (genebank) about fifty sequences of uncultured Glomus spp. clones can be found with a very high similarity (99%) to the sequence of the SYMBORG strain investigated here. It seems therefore, that the new taxon has a cosmopolitan distribution and a wide host range. The most similar sequences were retrieved from samples from Japan (Ogura-Tsujita et al., 2013; Yamato et al. 2011), New Zealand (Russel & Bulman 2005), Africa (Merckx & Bidartondo, 2008) and from North America (Appoloni et al. 2008).”

Comment: While allegedly a cosmopolitan variety, the applicant does not supply evidence to substantiate this distribution. Sequence data is indicated as “similar” to that of other taxa, though the latter remain discriminated as separate species. Its host range is unknown, though alleged to be large. The New Zealand Glomus species referred to are 2 native species associated with a liverwort (Russell & Bulman 2005).

Comment: The applicant’s assessment of close relatives of G. iranicum var. tenuihypharum needs to be considered in light of the statement from the authors who described G. iranicum. Those authors wrote: “G. iranicum has no apparent molecular relatives among described Glomus spp.” https://www.tandfonline.com/doi/full/10.3852/09-302

Section 5 - Potential beneficial effects:

“Moreover, the presence of several variants of Glomus sp. in New Zealand was reported recently (Johansen et al., 2015) in a study to identify the presence of mycorrhizal fungi in the coastal and sand dunes areas, indicating that the presence of AMF is beneficial for maintaining plant vigour and for the dune restoration efforts.” Comment: NZ’s native arbuscular mycorrhizal fungi (AMF) are beneficial and important. Introduction of exotic species may result in competition with and/or displacement of native species.

Section 5 - Potential adverse effects:

“We should consider that arbuscular mycorrhizal fungi dated from 450 million years ago and had followed the natural evolution of most of the plant species until today. So, the use of Glomus iranicum var. tenuihypharum do not cause diseases, do not stablish parasitic relationships nor become a vector for animals or plants.”

Comment: Natural evolution of native AMF and native plants is positive, but introduction of exotic AMF to a new ecosystem does not bring guaranteed overall advantage.

“In conclusion, the introduction of Glomus iranicum var. tenuihypharum in New Zealand do not pose any risks to the environment and human health and safety neither to 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. In fact, the introduction of Glomus iranicum var. tenuihypharum will contribute to improve plant growth, the soil characteristics and recolonization of the soil by native species by providing better soil conditions, which will provide a sustainable environment, meeting the principles of the Treaty of Waitangi and satisfy the relationship of the Māori to the environment.”

Comment: Statements concerning impact on the NZ environment cannot be tested. For example, the following statement cannot be verified: “… neither to 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”.

Section 5 - Potential to harm native plants and to displace native soil microbes

“According to literature, there is no reference to any negative effects regarding the introduction of AMF in the soil, displacement of native species within the natural habitat nor causing deterioration of natural habitats. In this sense, the New Zealand’s inherent genetic diversity will remain unharmed. However, if there is any possible impact of the introduction of Glomus iranicum var. tenuihypharum on soil biodiversity, considering the soil treated and the limited dissemination capacity of Glomalean fungi, would be limited to a soil that had already an impoverished biodiversity. Furthermore, the application of Symborg's products is indicated to drip irrigation systems or to seed coating, which limits even more the area of application of our Glomus. However, any problem associated with our Glomus, could be easily fixed with a soil's sanitation to eliminate the Glomus.”

Comment: Use of the product cannot be limited to only soils with impoverished biodiversity. Sales of Symborg’s products such as MycoUp could occur beyond farming to include nurseries and restoration activities in native ecosystems.

Comment: It is important to also consider the composition of the whole product. By weight, the fungal inoculum in the product makes up 0.05% to 4.0% (see patent details below), meaning that 96.0 to 99.95% of the product comprises 2:1 smectite clays as a carrier. What is the biodiversity of this clay material? Earlier experience with a rival imported mycorrhizal inoculum product in the NZ market place yielded a range of viable fungi and bacteria (identities not determined) as contaminants of the carrier material. These organisms are also introduced to NZ soils whenever that product is used. Does the Symborg product have viable spores of other species of fungi and bacteria? (See also Patent Claims 11, 13 below)

Section 6.4. Discuss if it is highly improbable, after taking into account the proposed controls, that the organism after release:

“…. Moreover, the eradication of Glomus iranicum var. tenuihypharum is as simple as removing the plant roots completely from the soil and an after-application of a fungicide to guarantee the elimination.”

Comment: Sales and application of Symborg’s product range is highly likely to be of a scale far beyond a small number of discrete plants, and hence removal of inoculated plant roots seems impracticable in the event that elimination of the fungus is required.

“Although the species Glomus iranicum has not been detected in New Zealand so far, it does not mean it is exotic since closely phylogenetic species has been found. As mentioned in most of the references, there is a lot of misidentification of microbial species worldwide, and the fact that our Glomus was never identified in New Zealand may be related with the lack of studies carried out in New Zealand or to misidentification of the species.”

Comment: The applicant suggests that G. iranicum could occur in NZ, although there has been no attempt (eg, by the applicant) to locate it. But the applicant is not here commenting on the subject of this application which is their patented var. tenuihypharum (of G. iranicum). Again, no work has been undertaken by the applicant to look for the relevant variety in NZ.

Composition of Patented Product

The following provides the Abstract and extracted Claims concerning Symborg’s patented product, based on patent information at https://patents.google.com/patent/WO2015000612A1/en. Of particular relevance are the following claims:

Claim 5: Composition includes 0.05% to 4% by weight of Glomus propagules. Ie, smectite clays and other materials constitute 96-99.95% of the product

Claims 9-14: Composition includes a range of biocidal chemicals and viable organisms. Are these chemicals (Claims 10, 12, 14) approved in NZ? Are all organisms incorporated as biofungicides and bioinsecticides (Claims 11, 13) approved for importation into NZ?

Claim 15: Composition includes a bio-stimulant bacterium “Pasteuria sp.” Is this a new organism?

“Abstract

Glomus iranicum var. tenuihypharum var. nov. strain deposited under BCCM deposit number 54871, comprising the sequence identified by SEQ ID NO: 1; composition comprising said strain and 2:1 smectite clays and use thereof as bio-stimulant. The invention also discloses a composition comprising said strain, fungicides, bio-fungicides, insecticides, bio-insecticides, nematicides and bio-nematicides.”

19 CLAIMS

1. - Glomus iranicum var. tenuihypharum var. nov. strain deposited under BCCM deposit number 54871, characterized in that it comprises the sequence identified by SEQ ID NO: l . 2. - Composition characterized in that it comprises a strain of Glomus iranicum var. tenuihypharum var. nov. deposited under BCCM deposit number 54871 comprising the sequence identified by SEQ ID NO: 1 and 2: 1 smectite clays. 3. - Composition according to claim 2, characterized in that said 2: 1 smectite clays are dioctahedral or trioctahedral. 4. - Composition according to claim 2, characterized in that said 2: 1 smectite clays are selected from the group consisting of sepiolite, attapulgite, nontronite and saponite. 5. - Composition according to one of claims 2 or 3, characterized in that the concentration of said Glomus iranicum var. tenuihypharum var. nov. strain is between 0.05 and 4% by weight. 6. - Composition according to claim 5, characterized in that said concentration is between 0.1 and 3% by weight. 7.- Composition according to any of claims 2 to 6, characterized in that the form of presentation of said composition is powder, emulsifiable concentrate or granules. 8. - Composition according to any of claims 2 to 7, characterized in that said composition is a liquid, a solid or a gel. 9. - Composition according to any of claims 2 to 8, characterized in that it comprises at least one fungicide, at least one bio-fungicide, at least one insecticide, at least one bio-insecticide, at least one nematicide and/or at least one bio-nematicide. 10.- Composition according to claim 9, characterized in that said fungicide is 20 selected from the group consisting of Maneb, Mancozeb, Metalaxyl-Ridomil, Myclobutanil, Olpisan, Propamocarb, Quintozene, Streptomycin, Sulfur, Thiophanate- methyl, Thiram, triforine, Vinclozolin, Zinc white, Zineb, Ziram, Banrot, Fixed copper, Chlorothalonil, Chlorothalonil, Captan, Chloroneb, Cyproconazole, Zinc ethelene, bisdithiocarbamate, Etridiazole, Fenaminosulf, Fenarimol, Flutolanil, Folpet, Fosetyl- AL and Iprodione. 11. - Composition according to one of claims 9 or 10, characterized in that said fungicide is selected from the group consisting of Trichodermas sp, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, Streptomyces sp, Coniothyrium minitans and Pythium oligandrum. 12. - Composition according to any of claims 9 to 11, characterized in that said insecticide is selected from the group consisting of organophosphate, carbamate and neonicotinoid. 13. - Composition according to any of claims 9 to 12, characterized in that said bio- insecticide is selected from the group consisting of Bacillus sp. Chromobacterium sp., Beauveria sp. and Metarhizium sp. 14. - Composition according to any of claims 9 to 13, characterized in that said nematicide is organophosphate or carbamate. 15. - Composition according to any of claims 9 to 14, characterized in that said bio-stimulant is Pasteuria sp. 16. - Method for obtaining a composition according claims 2 to 15, characterized in that it comprises: (a) coating inoculation of a seed of a host plant with the Glomus iranicum var. tenuihypharum var. nov. strain deposited under BCCM deposit number 54871, (b) cultivating said plant in watering cycles of between 7 to 10 days on a reproduction substrate comprising smectite clays in a percentage above 52% of the total weight of said substrate, (c) discontinuing said watering for a period equal to or greater than 20 days, (d) removing the aerial part of the plant and removing the substrate and 21 (e) milling said substrate below 80 microns at a temperature between 25 and 30° C to obtain said composition. 17. - Use of the composition according to any of claims 2 to 16 as bio-stimulant. 18. - Use according to claim 17, characterized in that said composition is applied to the plant by means of seed treatment, root treatment, roots embedded in an emulsion, addition to irrigation water, irrigation, application of powder to the root system or application of emulsion injected into the root system.

Composition of Symborg’s product MycoUP

The following provides data for Symborg’s product MycoUP – extracted from: https://s3-us-west-1.amazonaws.com/www.agrian.com/pdfs/MycoUp_MSDS.pdf

Section 1 - on the concentration of Glomus spores in the product (12,000 propagules per 100 mls); Section 13 - warning about spread into waterways

………………….

SUBMISSION 127409

From: submissions Subject: FW: APP203514 Application Open for Public Submissions - Soil fungus

Dear EPA

Thank you for the opportunity to comment on APP203514 - to release the arbuscular mycorrhizal fungus Glom us iranicum var. ten uihyph arum in New Zealand .

The Department of Conservation is strongly opposed to this proposal because of the extremely broad host range of the fungus and the likelihood that it will impart substantially increased vigour to a wide range of weeds as well as pasture and crop plants. Benefits to crop host plants as taxonomically diverse as maize, Viburnum, capsicums, strawberries, lettuce, grapes, tomatoes and melons are discussed in the application and in this paper: https://www.researchgate.net/publication/322571222 Application of Arbuscular Mycorrhizae Glomus it anicum var_ tenuihypharum var_nova_in_Intensive Agriculture A Study Case. These plants span the taxonomic diversity in the angiosperms as a whole; therefore this fungus could potentially colonise any angiosperm (flowering plants in the broadest taxonomic sense). This makes concerns about weediness very real.

It also has the potential to widely destabilise ecological balances in natural and semi-natural ecosystems by altering the relative vigour of different species and providing some with a new competitive advantage. It has the potential to turn formerly benign plants into significant weeds. This applies to agricultural weeds as well as those of ecological concern. The fungus may well rebound to the detriment of farmers as well as the broader community, and end up requiring substantial new applications of herbicides across the landscape.

We understand that the fungus can also act as a nematicide. Whereas in agricultural and horticultural situations nematodes are sometimes pests, in natural ecosystems where nematodes and related organisms are a natural component the fungus may have adverse impacts on the soil ecosystem and consequently have wider ecological impacts.

Glomus iranicum tenuihypharum is particularly associated with saline soils in its native habitat. Natural saline ecosystems are under particular pressure in New Zealand through conversion to farmland, being affected by surrounding land management, and invasion by weeds, and are listed as a Critically endangered ecosystem (Holdaway et al. 2012; https://www.landeareresearch.co.nz/publications/factsheets/rare- ecosystems/inland-and-alpine/inland-saline-salt-pans). Additional pressure from weeds made more robust and saline tolerant by this fungus would compound the problems faced by these threatened ecosystems, by leading to the displacement of native species from saline ecosystems through weed competition. Estuary vegetation such as salt marsh is also threatened by weeds in some areas.

We acknowledge that this fungus is not wind-dispersed and therefore spread will be slow. However, long- range spread is still possible via burrowing/soil disturbing animals such as rabbits and particularly pigs, and in soil on boots and machinery. The fact that the impact may occur sometime in the future doesn't make it any less of a concern. If the benefits to crop vigour are as great as the application claims, the fungus is likely to be introduced on farms all over the country, so have an extremely wide platform to invade native ecosystems from. Kauri die-back, Phytophthora agathidicida, provides a perfect example of a pathogen dispersed only by water or soil movements, yet having severe and widespread impacts in natural ecosystems right now, with tracks being closed to avoid further spread by people.

The applicants state that the fungus could be removed by removing the host. While this could be achieved in some situations by sterilisation of soil and plants, this could only apply on a very local scale. This fungus 1 looks like it is intended to be used across a wide range of plant systems including vine yards, plant based grey water management systems, horticultural systems, and glasshouses.

This application raises numerous questions for us, including:

• What is the impact of this fungus on native species and complex ecosystems overseas?

• Do all plant species respond similarly?

• Will the fungus distort native ecosystems by altering the balance between native species through altering competitive abilities?

• How do serious saline ecosystem pests such as spartina and saltwater paspalum respond to the fungus?

We also concur with the following concerns raised in the submission of Peter Buchanan (Manaaki Whenua — Landcare Research):

• Likelihood of dissemination in the environment

• Risks from new species associations with introduced species are quite different from those in coevolved native species

• Impossibility of limiting application to impoverished soils

• Risks of other, hitch-hiker organisms in the commercial preparation

• Uncertainty about the degree of relatedness between Glomus iranicum var. tenuihypharum and other Glom us species already present in New Zealand, and concern that no attempt has been made to fill this knowledge gap.

Should there be a hearing on this application, the Department of Conservation would wish to present evidence at the public hearing.

Yours faithfully

Rod Hitchmough Scientific Officer (Biosecurity) Department of Conservation—Te Papa Atawhai National Office, PO Box 10 420, Wellington 6143 18-32 Manners St, Wellington 6011 Cell: 027 408 3481 T: +64 4 471 3249 F: +64 4 381 3057 www.doc.govt.nz

2 SUBMISSION 127410

SUBMISSION FORM For Hazardous Substance and New Organism Applications

Once you have completed this form Send by post to: Environmental Protection Authority, Private Bag 63002, Wellington 6140 OR email to: [email protected]

Submission on application APP203514 number: Name of submitter or contact for Dr Cliff Mason joint submission: Organisation name N/A (if on behalf of an organisation): Postal address: 37 Ocean View Road Norhtcote AUCKLAND 0627

Telephone number: (09) 480 5756

Email: [email protected]

I wish to keep my contact details confidential

The EPA is likely to post your submission on its website at www.epa.govt.nz. We also may make your submission available in response to a request under the Official Information Act 1982.

www.epa.govt.nz 2

Submission Form

I support the application

I oppose the application

I neither support or oppose the application

The reasons for making my submission are1: (further information can be appended to your submission, see footnote). 1) General concern regarding the introduction of any alien organism into the New Zealand ecosystem. 2) Special concerns regarding the introduction of a microorganism. 3) Special concerns regarding the introduction of an organism into the environment of the soil. 4) Special concerns regarding the introduction of an arbuscular mycorrhizal fungus (AMF) Expanding upon these in turn: 1) The intentional introduction of an alien organism is a potentially irreversible act with potentially major and even catastrophic consequences. Because of these, any introduction should only occur in the situation of a pressing need for the introduction and an inability to meet the need by any other practicable means, and with assurance, as far as may be reasonably ascertained, that there are negligible risks to any valued system or service. In the present Application, the need is clearly lacking, the sole motivation for the Application being commercial. The assessment of risks is severely compromised by a major lack of specific knowledge of soil ecology in general and of the role of AMFs in ecological processes. The knowledge that we have serves only to clearly indicate that our ignorance is much greater. This unusually overt lack of relevant knowledge by which to assess either benefits or risks, combined with the absence of any demonstrated need fot the proposed introduction, should disqualify the Application on first principles. 2) Microorganisms present intrinsic difficulties in containment and identification in the environment which, combined with rapid growth and/or spread make them liable to be the cause of damage which is imperceptible until irreparably advanced. They are often involved (as is very much the case with AMFs) in fundamental ecological processes and often operate at the interface between larger organisms and between these and the abiotic environment. As a result, serious disturbances in microbiological function through the displacement or inactivation of pre-existing microbiota or the activity of the new microorganism can result in ecosystem-wide effects of an extreme nature and scale. 3) The soil is a cryptic environment that has been very difficult to investigate. Only recently have specialised techniques made it possible to gain some information about the composition and functioning of soil communities, especially the microbiological components. This information remains very preliminary and little has been integrated into wider understanding of ecosystems. In New Zealand, the situation has been dire due to a critical lack of soil scientists and it is likely that the expertise to properly assess the Application is unavailable. Problems inherent in soil microbiology have been made obvious recently in the case of phytophthera infection of kauri. 4) AMFs epitomise the fundamental importance of microorganisms in local and global ecology and also exemplify the irreducible complexity of the ecological processes involved. These are too extensive to detail in a submission but are well covered in a review article (A. Wiilis, B.F.Rodrigues and P.J.C. Harris, 'The Ecology of Arbuscular Mycorrhizal Fungi'; Critical Reviews in Plant Sciences 32:1, 1 - 20). Some of the relevant matters, most of which are only very sketchily covered or are omitted entirely from the Application, are outlined with appropriate references below: The taxonomy of AMFs is controversial and there is evidence of cryptic speciation and other forms of biological diversity that are not reflected in the present taxonomic tables. In short, there are more forms of AMF extant than we have any formal knowledge of. (See e.g. 'Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management;

July 2016 EPA0190 3

Submission Form

http://dx.doi.org/10.5772/58231 page 164; 'Arbuscular mycorrhizal fungal communities exposed with new DNA sequencing approach", https://phys.org/print425901911.html) There is evidence of ongoing evolutionary change and adaptation even in this ancient and apparently very stable group of organisms (See Daniel Croll and Ian R Sanders, 'Recombination in Glomus intraradices,a supposed ancient asexual arbuscular mycorrhizal fungus',BMC Evolutionary Biology, 2009, 9:13). Added to the paucity of informaton related to NZ AMFs, these indicate the inadequate state of knowledge to ensure any reasonable degree of safety in introducing an alien AMF to NZ. The addition of inocula of AMFs have highly variable effects on resident AMF populations with documented cases of detrimental effects both on the native species and on the collective ecosystem services of the AMF community (See e.g. M. Janouskova et al; 'Effects of Inoculum Additions in the Presence of a Preestablished Arbuscular Mycorrhizal Fungal Community', Applied and Environmental Microbiology October 2013, 79 (20); pp6507 - 6515) Other risks are detailed in the attached references: Schwartz et al 'The promise and the potential consequences of the global transport of mycorrhizal fungal inoculum' Ecology Letters (2006) 9: 501 - 515 Miranda M Hart et al 'Fungal inoculants in the field: Is the reward greater than the risk?', Functional Ecology 2018 32:126 - 135 It is also clear that the benefits from the use of AMF inoculation is very dependent upon a large number of concurrent factors such that results from the use of AMFs vary widely from time to time and place to place. The achievement of optimum benefit requires simultaneous management of these many factors and the outcome will still be uncertain. New Zealand also has relatively little of the depauperate soils that are found in other countries with a long history of intensive agriculture that show the most benefit from use of AMFs. Rather than introducing a novel AMF into the NZ environment, a better course of action - with the possibility of achieving the proposed benefits of the Application without the inherent risks - would be to further research the state of AMFs that exists in our agricultural and other soils and the possibility of managing these for agricultural and biodiversity purposes. To reiterate; there is no apparent need for the introduction of this AMF, there is unlikely to be any significant benefit from introduction, there are apparent significant risks from such an introduction, inter alia, to native biodiversity (both native AMFs and all species within the ambit of the agents colonisation capabilities and within ecosystems in which it is established). It is also impossible in the present state of knowledge, and in the foreseeable future given the extreme complexity and random nature of relevant ecosystemic matters, to make a meaningful assessment of the risks and benefits. In this situation, the only defensible course of action is to reject the Application.

July 2016 EPA0190 4

Submission Form

I wish for the EPA to make the following decision: Reject the Application.

July 2016 EPA0190 Effects of Inoculum Additions in the Presence of a Preestablished Arbuscular Mycorrhizal Fungal Community

Martina Janoušková,a Karol Krak,a Cameron Wagg,b Helena Štorchová,c Petra Caklová,a Miroslav Vosátkaa Institute of Botany, Academy of Sciences of the Czech Republic, Pru˚honice, Czech Republica; Ecological Farming Systems, Agroscope Reckenholz Tänikon Research Station, Zürich, Switzerlandb; Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republicc

Communities of arbuscular mycorrhizal fungi (AMF) are crucial for promoting plant productivity in most terrestrial systems, including anthropogenically managed ecosystems. Application of AMF inocula has therefore become a widespread practice. It is, however, pertinent to understand the mechanisms that govern AMF community composition and their performance in order to Downloaded from design successful manipulations. Here we assess whether the composition and plant growth-promotional effects of a synthetic AMF community can be altered by inoculum additions of the isolates forming the community. This was determined by following the effects of three AMF isolates, each inoculated in two propagule densities into a preestablished AMF community. Fungal abundance in roots and plant growth were evaluated in three sequential harvests. We found a transient positive response in AMF abundance to the intraspeciﬁc inoculation only in the competitively weakest isolate. The other two isolates responded negatively to intra- and interspeciﬁc inoculations, and in some cases plant growth was also reduced. Our results suggest that increasing the AMF density may lead to increased competition among fungi and a trade-off with their ability to promote plant productivity.

This is a key ecological aspect to consider when introducing AMF into soils. http://aem.asm.org/

t is an ongoing objective in ecology to understand the mecha- tential effects on plant growth (18–20). Yet, little is currently Inisms that shape the community structure and productivity of known about the interactions among different AMF colonizing ecosystems, in order to ultimately maintain the services ecosys- one root system and the factors responsible for the presence and tems provide. Thereby, soil communities belowground are known abundance of particular AMF taxa among roots from the pool of to be a key element in maintaining the productivity and diversity propagules available in the soil. of communities aboveground (1, 2). Arbuscular mycorrhizal Here we assess the responses in both the mycorrhizal fungal fungi (AMF) are a guild of soil organisms that are dependent upon partners and the plant host when additional AMF propagules are on November 27, 2018 by guest plant hosts to acquire carbon and that provide in exchange many introduced into a preestablished AMF community of three Glo- services for the plants, such as improving their nutrient acquisi- maceae isolates. We thus mimicked the approach of inoculation tion, productivity, coexistence, and pathogen protection (3). with native AMF (17) and changed the relative infectivity of these Considering the large potential of these symbiotic fungi to con- isolates in the soil. We expected that additional AMF propagules tribute to the ecological sustainability of managed ecosystems, would increase the abundance of the inoculated fungus and sup- efforts are being made to improve the resource use efficiency of press the development of the other two fungi. Second, we assessed arable and degraded soils by introducing AMF inocula. However, whether the AMF inoculation could improve plant growth-pro- there are many questions remaining regarding the conditions un- motional effects of the AMF community. In order to test this, we der which introduction of AMF into soils is successful at improv- grew medic (Medicago sativa L.) in soil with a previously estab- ing the plant growth-promotional effects of AMF communities lished AMF community or in the same soil after it had been ster- (4). The current challenge for improving soil productivity by AMF ilized. The three fungal isolates were introduced at different prop- community manipulations is in understanding the ecological agule amounts into both background treatments, and the systems constraints, such as competitive and complementary interactions were sampled at 6, 12, and 24 weeks to assess both fungal devel- with AMF genotypes present in the soil (5). opment and plant growth. AMF differ in their life traits and nutrient-foraging strategies (6, 7). These differences can be the basis for the complementary MATERIALS AND METHODS effect of greater AMF richness (8) or may favor the more-benefi- Experimental system. The experiment was based on a ruderal soil-fungi cial partners under the given conditions (9, 10). On the other system of a freshly leveled spoil bank of a surface mine near Chomutov, hand, AMF colonizing a root system compete for space and the North Bohemia, Czech Republic (see reference 21 for a site description). plant-derived carbohydrates (11), with potential trade-offs with the beneficial effects of the symbiosis with the host plants (12–14). Understanding the interactions among coexisting AMF is there- Received 28 June 2013 Accepted 10 August 2013 fore important not only from the point of view of basic commu- Published ahead of print 16 August 2013 nity ecology, but also for predicting the effects of AMF community Address correspondence to Martina Janoušková, [email protected]. manipulations, such as when AMF are inoculated into soils con- Supplemental material for this article may be found at http://dx.doi.org/10.1128 taining AMF communities. Inoculation may decrease AMF diver- /AEM.02135-13. sity in roots, resulting in both positive and negative plant growth Copyright © 2013, American Society for Microbiology. All Rights Reserved. responses (15–17). Shifts in propagule densities of AMF in soil can doi:10.1128/AEM.02135-13 affect the quantitative composition of the communities, with po-

October 2013 Volume 79 Number 20 Applied and Environmental Microbiology p. 6507–6515 aem.asm.org 6507 Janoušková et al.

The cultivation substrate was prepared by mixing homogenized gray mio- The soil-sand cultivation substrate described above was sterilized by cene clay collected on the spoil bank surface with sand, in the ratio of 1:2. ␥-irradiation and used to ﬁll 33 pots, each 10 liters in volume. Every pot

The mixture had the following main parameters: pH (H2O), 7.5; Corg, was inoculated with 230 IP of each fungus, which corresponded to 1.6 g of Ϫ1 1.15%; N, 0.06%; Olsen-P (0.5 M NaHCO3 extractable), 2.93 mg kg . G. claroideum and G. intraradices and 50 g of the G. mosseae inoculum. Two of the three fungal isolates used originated from the same spoil The mixed inoculum was prepared for each pot separately by weighting bank as the clay substrate: Glomus claroideum Schenck & Smith Chomu- and thoroughly mixing the air-dried culture substrates of known inocu- tov (isolate referred to herein as G. claroideum) and G. intraradices sensu lation potentials. The inoculum was placed into the center of the pot, lato Chomutov (isolate referred to herein as G. intraradices; for closer about 3 cm below the surface, and the pot was planted with eight pre- specification, see reference 22). The third isolate used, G. mosseae (Nicol. germinated medic plantlets. The pots were cultivated in a glasshouse with & Gerd.) Gerd. & Trappe BEG95 (isolate referred to herein as G. mosseae), a light supplement (12 h, metalhalide lamps, 400 W) and fertilized once in originated from another spoil bank near Most, in the same geographic 2 weeks with 200 ml of the P2N3 nutrient solution (30). region (labeled G. mosseae ALB in reference 23). After 6 months of cultivation, the shoot biomass was cut. From the Several cultures were established for each isolate in a mixture of zeolite center of each pot, a root sample was extracted using a soil corer and wet and sand (1:1) with medic (Medicago sativa L. cv. Vlasta) as the host plant, sieving. Half of the obtained root samples were immediately frozen in Downloaded from using 200 spores collected from a pooled soil sample from three multi- liquid N, stored at Ϫ80°C, and later used for the determination of copy spore cultures. The established cultures were used for the characterization numbers (CN) of each fungus (as described below), and another portion of genetic diversity and as the source of inoculum. was used for a check of root colonization after trypan blue staining (31). Design and optimization of quantiative PCR (qPCR) assays. The The substrates from the pots were air dried and homogenized, including genetic variations of the studied isolates in the large subunit of the nuclear most of the fine roots; thick roots were removed in order to decrease the ribosomal DNA (nrDNA LSU) were characterized in detail as described amount of root biomass in the homogenized substrate. The resulting sub- for the isolate G. intraradices in reference 22. The obtained sequences were strate was subsequently termed the AMF substrate and contained a syn- aligned together with sequences deposited in GenBank under the respec- thetic AMF background community. Its infectivity was determined by the tive species names by using Clustal X (24), and the alignments were cor- MPN method as described above, together with determination of the rected manually in BioEdit (25). Initially, alignments for the individual infectivity of the inocula used later in the experiment. The inoculation http://aem.asm.org/ species were done separately and subsequently combined into a final potential of the established AMF community was 22 IP mlϪ1. The fre- alignment. Maximum parsimony analyses were performed based on both quency of root colonization as obtained from 10 randomly selected root the individual as well as the complete sequence alignments by using samples was 84% (Ϯ2% standard error of the mean [SEM]). The G. in- PAUP* (26) (see Fig. S1 in the supplemental material). traradices isolate was present in the roots with the highest nrDNA copy Primers discriminating between the studied isolates were designed for numbers (on average, 120 ϫ 103 CN ngϪ1 isolated DNA, Ϯ28 ϫ 103), G. claroideum and G. mosseae (see Table S1 in the supplemental material) followed by G. claroideum (3 ϫ 103 CN ngϪ1, Ϯ0.57 ϫ 103), while G. based on the complete sequence alignment obtained using Primer 3 Plus mosseae was almost absent. It was detected in very low copy numbers (50 (27). Variations of nrDNA LSU sequences in the G. mosseae isolate pre- and 276 CN ngϪ1 isolated DNA) in only two out of seven analyzed root vented the design of a single primer pair that would amplify all the se- samples. quence variants of this isolate but not cross-amplify the other fungal spe- Half of the substrate was then sterilized by ␥-irradiation to be used as on November 27, 2018 by guest cies. Two specific primer pairs were therefore developed, each amplifying control substrate without AMF. only a part of the G. mosseae ribotypes, and applied as a duplex assay. For Establishment and cultivation of the experiment. Seven inoculation the G. intraradices isolate, previously designed primers (22) were used, as treatments were established, one each in the AMF substrate and the con- they were found suitable for the present study. The specificity of these trol substrate (14 treatments in total). The treatments were replicated 24 qPCR assays was tested in silico using the Fast PCR software (28) as well as times, to be harvested at three consecutive harvests (eight replicates per experimentally by cross-amplification experiments using different tem- harvest). This resulted in 336 pots in total, each 0.7 liters in volume and plates (plasmid standards, medic DNA extracted from roots and leaves, planted with one medic plant. The inoculation treatments were as follows: fungal DNA extracted from spores, and DNA extracted from medic roots (1) noninoculated; (2 and 3) inoculated with G. claroideum at two inoc- colonized by each fungal isolate). The preparation of plasmid standards, ulum levels; (4 and 5) inoculated with G. intraradices at two levels; (6 and qPCR, and the estimate of amplification efficiencies followed in general 7) inoculated with G. mosseae at two levels (see Fig. 1 for an overview). the procedures described in reference 22. The details on annealing tem- The inocula were prepared and standardized as described above based peratures, primer concentrations, amplicon lengths, and the estimated on the MPN method. Again, the inocula of G. claroideum and G. intrara- amplification efficiencies for each qPCR assay are summarized in Table S1 dices had higher infectivities (81 and 57 IP mlϪ1, respectively) than the G. of the supplemental material. mosseae inoculum (4 IP mlϪ1). The inoculation level of 150 IP per pot, Precultivation of the synthetic AMF community. The three AMF applied for all three isolates, was selected as on the order of magnitude isolates were cultivated together to establish a background AMF commu- corresponding with the recommendations of inoculum producers for in- nity. In order to give them the same chance to develop within the com- oculation. A higher inoculation level of 400 IP per pot was applied with G. munity, the mycorrhizal inoculation potential of the initial inoculum was claroideum and G. intraradices, but this level could not be applied with G. standardized to equal numbers of infective propagules (IP) of each isolate. mosseae because of the low infectivity of the G. mosseae inoculum. Instead, To achieve this, the substrates from five 6-month-old cultures of each the remaining G. mosseae inoculum was used to establish at least a low isolate were checked microscopically for the presence of spores and ab- inoculation level of 20 IP per pot. sence of contamination, homogenized, and air dried. Five serial dilutions The inocula were weighted for each replicate separately and mixed of the inocula with the ␥-irradiated experimental substrate (1:10 to 1:105, with sterilized zeolite-sand mixture to constant volumes corresponding to vol:vol) were planted with medic in five replicates. When the roots of the the highest volume of inoculum added (38 ml in the G. mosseae 150-IP plants had grown through the whole soil volume of 85 ml (after 5 weeks), treatment). Seedlings of medic were germinated in autoclaved sand and the presence and absence of root colonization in the root systems was precultivated for 3 weeks in the sterilized experimental substrate. They scored and used to calculate the number of infective propagules by the were inoculated at planting into the experiment by an inoculum layer most probable number (MPN) method (29). The G. claroideum and G. inserted about 3 cm below the surface. In order to equalize the microbial intraradices inocula had an identical inoculation potential of 150 IP mlϪ1 community compositions with the different treatments, all pots were ir- of substrate, while the inoculation potential of G. mosseae was distinctly rigated with bacterial filtrate from the AMF substrate and the inoculum. lower (4 IP mlϪ1). The filtrates were obtained by shaking a sample of the AMF substrate

6508 aem.asm.org Applied and Environmental Microbiology AMF Inoculation into a Background Community

AMF community relative to the intensity when it is the only AMF present. This response to a background AMF community was calculated as fol- ϭ Ϫ ϩ lows: RIIcommunity (O M)/(O M), where O was the observed abundance of an AMF in roots when a background AMF community was present and M was the mean of the same AMF detected in roots in the absence of a background community of the same IP level and harvest

period combination. We tested whether the RIIcommunity of each AMF was inﬂuenced by the harvest period and the IP level at which the fungi were inoculated by using a two-way ANOVA. The RII metric was also used to assess the response of AMF to the

addition of inter- and intraspeciﬁc inocula (RIIinoculation). In this case, O was the observed abundance of an AMF when the AMF inoculum was added and M was the mean abundance of a fungus detected in roots in the

background AMF community with no additional AMF inoculum added. Downloaded from This RII measure was assessed for variations among AMF identity, the IP FIG 1 Schematic of the experimental design. The precultivation stage ren- level at which the fungus was inoculated, and the harvest period, by using dered the substrate colonized by a synthetic community of three arbuscular a three-way ANOVA. All model means were compared to an RII of 0 to mycorrhizal fungal isolates (AMF substrate) and sterilized substrate without test for a significant response in the AMF abundance within the host plant AMF background (control substrate). Each treatment of the experiment is roots. represented by one small pot in the lower part of the diagram. Besides nonin- Analyses of plant performance. All biomass data were square-root oculated treatments (empty pots), each of the three isolates of the community transformed before analyses to improve homoscedasticity. Separate was inoculated with 150 IP (middle-sized geometric symbols). Additionally, ANOVAs for each harvest and AMF community background level were the G. claroideum and G. intraradices pots were inoculated with 400 IP (large used to test for differences in shoot and root biomass between the noni- circles and triangles) and the G. mosseae isolate was inoculated with 20 IP (small squares). For specification of the isolates, see Materials and Methods. noculated control treatments and each of the AMF-inoculated treat- http://aem.asm.org/ ments. In order to determine whether the IP level (150 IP or an alternative level) and the presence of a background AMF community influenced the growth-promotional effect of an AMF inoculum, separate two-way (about 500 g) and samples of the inocula (about 200 g each) overnight in ANOVAs for each AMF species, with the IP level and the background as distilled water, passing the resulting soil suspensions twice through a filter sources of variation, were used. paper (Whatman no. 1), and adjusting the volume to that required. The Additionally, the RII was used to calculate the response of total plant filtrate from the AMF substrate was added into every pot with control biomass to AMF inoculum additions into the AMF background. Here, M substrate (10 ml each). The filtrates from the inocula were mixed together was the mean total biomass of plants grown with a background AMF and added into every experimental pot (10 ml each). The experimental community, and O was the observed biomass produced when additional pots were cultivated in a glasshouse with a light supplement (12 h, metal- AMF inoculum was added. This plant response index was assessed for on November 27, 2018 by guest halide lamps, 400 W) and fertilized once in 2 weeks with 50 ml per pot of variations among identities of the fungal species inoculated, the IP level at P2N3 nutrient solution (30). which the fungus was inoculated, and the harvest period, by using a three- Harvest and data collection. After 6, 12, and 24 weeks, eight pots per way ANOVA. treatment and harvest were destructively harvested. At each harvest, root For all ANOVAs, nonsignificant interaction terms were removed to systems were carefully washed from the substrate, weighed, and cut into capture the full amount of variation explained by the main effects. How- 1-cm segments. A subsample of 100 mg (fresh weight) was immediately ever, full ANOVA results are presented in Tables S3 and S4 in the supple- frozen in liquid N and stored at Ϫ80°C. Another part was used for micro- mental material to show the magnitudes of these interaction effects. All scopic determination of root colonization after staining with 0.05% data manipulations and statistical analyses were performed in R for Mac trypan blue in lactoglycerol (31). The remaining roots and the shoots were OS X version 2.15.1 (R Foundation for Statistical Computing). dried at 80°C for 24 h. Nucleotide sequence accession numbers. Representative sequences Shoot and root dry weights were determined for the experimental of AMF nrDNA LSU were submitted to GenBank under the accession plants. Root colonization was evaluated by microscopy (Olympus BX60, numbers KC522414 to KC522421. 100ϫ magnification) according to the methods described in reference 32. The intensity of colonization of the root system was determined using the RESULTS program Mycocalc (http://www.dijon.inra.fr/mychintec/Mycocalc-prg Root colonization. Root colonization by AMF was not observed /download.html). Genomic DNA was extracted from the root samples by using a DNeasy in any replicates at any harvest where there was no background plant minikit (Qiagen) according to the manufacturer’s instructions. AMF community present and no addition of AMF inoculum. DNA extracts from root samples were quantified spectrophotometrically, When AMF were inoculated into the control background (no pre- and 10 ng of total genomic DNA was used as the template for qPCR, which vious AMF present), colonization intensity varied among the ϭ was performed as described in reference 22. Six replicate root systems per AMF species inoculated, depending on the time of harvest (F4,89 treatment and harvest were analyzed. All target sequences were quantified 28.0; P Ͻ 0.0001) (Table 1). At 6 weeks, root colonization was in all root samples. lower for G. mosseae than for G. intraradices or G. claroideum.At Analyses of fungal root colonization. Root colonization parameters 12 weeks, both G. mosseae and G. claroideum resulted in signifi- were assessed by an analysis of variance (ANOVA) for variations among cantly lower colonization intensities than G. intraradices, while at harvest period, the identity of the AMF inoculated, and the IP level, and by 24 weeks all three fungi differed from each other (G. claroideum Ͻ a three-way ANOVA separately for plants with and without a background Ͻ AMF community present. The nrDNA copy numbers of each fungus were G. mosseae G. intraradices)(Table 1). Additionally, for the con- used to assess the response in the abundance of each AMF to the various trol background the colonization intensity also differed overall AMF inoculation treatments. This was done using the relative interaction between plants receiving 150 IP versus alternative IP levels, de- ϭ ϭ intensity (RII; see reference 33) to calculate an index reflecting the com- pending on the identity of the fungus inoculated (F2,95 4.61; P petitive ability of AMF to colonize roots in the presence of a background 0.01). Inoculation of G. mosseae at 150 IP resulted in a greater

October 2013 Volume 79 Number 20 aem.asm.org 6509 Janoušková et al.

TABLE 1 Intensity of root colonization by AMF by week, AMF species, troduced AMF were less abundant in roots in the presence of a and IP background AMF community than when inoculated at the same Mean (SE) intensity of root levels in the control background (Fig. 2; see also Table S2 in the colonizationa supplemental material for raw means and standard errors of AMF nrDNA detection). The suppression was most pronounced for G. Week of AMF No. of Control AMF harvest inoculated IP background background mosseae. Additionally, the performance of G. mosseae only differed among harvests (F ϭ 9.16; P ϭ 0.0007; see Table S3 in the 6 None 0 62.0 (9.12) abc 2,32 6 G. mosseae 150 28.5 (13.8) ad, C 60.0 (16.0) abc supplemental material for full ANOVA results), where it was least 6 G. mosseae 20 7.30 (7.03) ad, B 67.5 (15.4) abc detected at 24 weeks relative to earlier harvests despite IP level 6 G. intraradices 150 48.0 (26.4) e, A 52.3 (15.4) ab (Fig. 2). For both G. intraradices and G. claroideum, the abundance 6 G. intraradices 400 50.5 (12.9) e, A 55.7 (8.33) ab response was generally smaller and depended upon the time of ϭ ϭ ϭ 6 G. claroideum 150 37.5 (10.5) bce, BC 56.2 (12.0) ab harvest and level of IP added (F2,27 3.76, P 0.04 and F2,30

6 G. claroideum 400 34.0 (4.60) bce, C 52.3 (11.5) ab 9.85, P ϭ 0.0005 for G. intraradices and G. claroideum, respec- Downloaded from 12 None 0 62.2 (12.4) abc tively). Only at 24 weeks did G. intraradices differ between IP 12 G. mosseae 150 34.5 (8.41) abd 59.5 (9.48) a levels, with 150 IP leading to a similar abundance as when no AMF 12 G. mosseae 20 13.0 (9.12) abd 40.7 (7.00) a background was present (Fig. 1). At 24 weeks, G. claroideum was 12 G. intraradices 150 89.3 (3.88) f 68.3 (4.84) abc the most suppressed in abundance despite IP level, while at 6 and 12 G. intraradices 400 88.2 (3.19) f 58.8 (9.52) abc 12 G. claroideum 150 33.7 (13.7) abc 59.7 (13.8) ab 12 weeks, it reached similar abundance as without AMF back- 12 G. claroideum 400 24.8 (4.17) abc 54.8 (7.41) ab ground either at 150 IP (6 weeks) or at 400 IP (12 weeks) (Fig. 2). 24 None 0 68.5 (21.1) abc AMF responses to additional inoculation. Both G. intraradi- 24 G. mosseae 150 44.0 (11.2) ce 71.8 (9.43) bc ces and G. claroideum varied in their responses to inoculum addi- http://aem.asm.org/ 24 G. mosseae 20 42.0 (17.3) ce 68.0 (8.22) bc tions (RIIinoc.), depending on the time of harvest, the identity of 24 G. intraradices 150 85.3 (10.8) f 80.2 (3.77) c the fungus inoculated, and the IP level at which the fungus was 24 G. intraradices 400 87.3 (8.48) f 72.0 (14.1) c ϭ ϭ ϭ ϭ inoculated (F4,86 2.55, P 0.04 and F4,86 3.64, P 0.009, 24 G. claroideum 150 12.8 (7.44) d 67.3 (10.6) bc respectively; see Table S3 in the supplemental material for full 24 G. claroideum 400 14.5 (11.8) d 69.2 (18.6) bc ANOVA results). Generally, the effects of inoculum addition were a Calculated according to the methods described by Trouvelot et al. (32). Means of the frequently negative (Fig. 3). same background community not sharing a common letter differed significantly (based The IP level of G. intraradices added did not influence its over- on Tukey’s honestly significant difference comparisons, P Ͻ 0.05). Lowercase letters (a to f) indicate differences among a harvest by interaction with the AMF species all relative abundance; however, at 400 IP, G. intraradices was inoculated. Capital letters (A to C) indicate differences among an IP level by interaction reduced in abundance relative to its abundance without addi- with the AMF species inoculated (only indicated at harvest 1). tional inocula at 6 and 12 weeks. G. intraradices was also sup- on November 27, 2018 by guest pressed by 400 IP of G. claroideum at 6 and 12 weeks, and by 20 IP of G. mosseae at 12 weeks, relative to its abundance without addi- colonization intensity than at 20 IP, while neither G. claroideum tional inocula. At 24 weeks, G. intraradices was not affected by any nor G. intraradices results differed significantly overall between IP inoculum additions. levels (Table 1). In the presence of a background AMF commu- G. claroideum was consistently reduced in abundance with the nity, the intensity of colonization only varied among harvests de- addition of 400 IP of intraspecific inoculum at all three harvests. ϭ ϭ pending on the AMF species inoculated (F2,102 3.04; P 0.02), Conversely, 150 IP of G. claroideum inoculum had no effect on G. as colonization intensity was greater at 24 than at 12 weeks for G. claroideum abundance after 6 weeks, but it had an increasingly mosseae-inoculated plants and greater at 24 weeks than at 6 weeks suppressive effect at the later harvests. Inoculation of G. intrara- for G. intraradices-inoculated plants (Table 1). dices at 150 IP marginally increased G. claroideum abundance at 6 AMF response to background community. In general, the in- and 24 weeks but reduced its abundance at 12 weeks. However,

FIG 2 Abundance of AMF in roots when a preestablished AMF background community is present relative to when AMF are inoculated in the absence of a background AMF community (RIIcommunity). This reflects the ability of AMF to achieve colonization levels in the presence of an AMF community relative to when no interspecific competition is present. The dotted black line indicates the colonization abundance when no background AMF community was present. Error bars indicate the 95% confidence intervals. Lines connecting means highlight the trend between harvests.

6510 aem.asm.org Applied and Environmental Microbiology AMF Inoculation into a Background Community Downloaded from http://aem.asm.org/ on November 27, 2018 by guest

FIG 3 The response for AMF abundance in roots to an additional AMF inoculum in the presence of a preestablished AMF background community (RIIinoc.). The dotted black line indicates the AMF abundance in roots achieved in the background AMF community when no additional AMF propagules were added. This reflects the influences of intra- and interspecific AMF densities on their abundance within roots. The dotted black line indicates the abundance of AMF when no AMF inoculum was added. Error bars indicate the 95% confidence intervals. Lines connecting means highlight the trends between harvests. with 400 IP of G. intraradices inoculated, G. claroideum was con- ground AMF community, only G. intraradices inoculated at 150 IP sistently suppressed in abundance at all three harvests. Inocula- after 12 weeks resulted in improved shoot biomass (Fig. 3). Inoc- tion with 150 IP of G. mosseae had no effect, but 20 IP of G. mosseae ulation with G. mosseae at 20 IP reduced shoot productivity, and increasingly suppressed G. claroideum in abundance at the later G. claroideum at 400 IP reduced both root and shoot productivity harvests. at 6 weeks (Fig. 4). The abundance of G. mosseae was only influenced by the iden- Plant performance differed in some cases, depending on the IP ϭ Ͻ tity of the AMF inoculated (F2,99 12.1, P 0.0001; see Table S3 level of inoculation (see Table S4 in the supplemental material for in the supplemental material). In general, intraspecific inoculum full ANOVA results). At both 6 and 12 weeks, G. mosseae-inocu- additions of G. mosseae increased its overall abundance within lated plants had a higher shoot biomass when inoculated with 150 roots relative to its background levels, whereas inoculation with G. IP than with 20 IP, despite the presence of an AMF background ϭ ϭ ϭ intraradices or G. claroideum significantly suppressed the abun- community (overall IP effects: F1,21 4.65, P 0.04 and F1,21 dance of G. mosseae in comparison with the noninoculated treat- 4.93, P ϭ 0.04, respectively). Additionally, at 24 weeks after inoc- ment (Fig. 3). ulation with G. claroideum at the higher 400-IP level, shoot bio- Plant performance. Overall plant biomass, both above- and mass was depressed in comparison to inoculation with G. claroi- belowground, increased with time and was generally greater in the deum at 150 IP in both control and AMF background treatments ϭ ϭ presence of a background AMF community (Fig. 4). Plant shoot (overall IP effect of G. claroideum: F1,21 4.90; P 0.04). The IP and root biomass were improved by the addition of AMF at all levels at which G. intraradices was inoculated did not significantly three harvests when no background AMF community was pres- alter shoot production in any case (see Table S4). Furthermore, ent. Only inoculation with G. mosseae at 20 IP did not significantly inoculation with 400 IP of G. claroideum reduced root biomass at ϭ ϭ ϭ improve root biomass at 6 and 24 weeks (P 0.12 and P 0.11, 6 and 24 weeks in comparison to the 150-IP inoculation (F1,21 ϭ ϭ ϭ respectively) relative to when no AMF were present in the control 6.79, P 0.02 and F1,21 5.15, P 0.03, respectively), despite the background. Conversely, comparing individual inoculated treat- background treatment. At 12 weeks, inoculation with G. intrara- ments to the uninoculated plants in the presence of the back- dices at the higher level of 400 IP improved root biomass in the

October 2013 Volume 79 Number 20 aem.asm.org 6511 Janoušková et al. Downloaded from

FIG 4 Means (with SE) for the biomass of roots and shoots grown in each inoculation treatment with and without an arbuscular mycorrhizal fungal background community (N, no inoculum added; M, G. mosseae isolate; I, G. intraradices isolate; C, G. claroideum isolate; AMF, with preestablished background community; control, without AMF background community). Shading indicates the different amounts of IP added of each isolate. Asterisks above/below means indicate http://aem.asm.org/ differences between the individual inoculated treatments means and the corresponding noninoculated treatment (N). When not indicated, the effect of inoculation was nonsignificant. absence of an AMF community, but it reduced root production in achieved in a previously established AMF community. Impor- the presence of an AMF community, resulting in an IP level by tantly, high levels of propagules of the inoculated fungus resulted ϭ ϭ background interaction (F1,20 10.7; P 0.003) (Fig. 3). The IP in lower abundance of the fungi in the roots. In some cases, this levels at which G. mosseae was inoculated did not significantly alter corresponded with a decrease in plant growth. root production in any case (see Table S4). A number of studies have demonstrated that the additional The response in total plant biomass to AMF inoculation host benefits following inoculation with multiple AMF are on November 27, 2018 by guest

(RIIinoc.) into the AMF background differed between the two IP levels likely context dependent (10, 34–36). Competition among ϭ ϭ overall (F2,95 14.4; P 0.0003). The 150-IP level generally resulted AMF for host resources may result in a trade-off with the host in greater plant performance than the alternative IP levels (Fig. 5). growth promotion abilities of the AMF community (12, 13). The response in total plant biomass also varied among a harvest by Our results support these previous ﬁndings in that increased ϭ ϭ AMF identity interaction (F4,93 3.38; P 0.01). Inoculation with G. AMF competition, here by increasing fungal density and/or intraradices at 24 weeks suppressed total biomass despite inoculation shifts in the established propagule balance, can affect the root level (Pϭ0.002), while the other two AMF did not vary greatly overall colonization process and decrease the promotion of host in response to AMF inoculum additions. growth by the AMF symbiosis. The performance of both the host plant and the AMF was dependent not only on the identity DISCUSSION of the AMF species inoculated, but also on the density of infec- Our results show that addition of AMF propagules results in tive propagules within the inoculum. Such results suggest that changes in the abundance of AMF away from the equilibrium state the addition of AMF propagules into established AMF commu-

FIG 5 The response in total plant biomass to an additional AMF inoculum when a preestablished AMF background community is present (RIIinoc.). The dotted black line indicates the biomass production when no additional AMF propagules were added. Error bars indicate the 95% conﬁdence intervals. Lines connecting means highlight the trends between harvests.

6512 aem.asm.org Applied and Environmental Microbiology AMF Inoculation into a Background Community nities can result in a trade-off with the ability of the AMF com- this dynamic with its progressive decline in nrDNA copy numbers munity to support the productivity of the plant host. as well as root colonization by the second and third harvests. Con- Inoculation effects on fungal development. The pool of sistent with the assumption of accelerated vitality peak and de- nrDNA copies of the AMF community was dominated by G. in- cline, the response of G. claroideum to intraspecific inoculation traradices, in accordance with observations of highly skewed nat- was progressively negative with time. In contrast to the negative ural communities being usually dominated by a single taxon (37). abundance response to intraspecific inoculation, the abundance However, nrDNA copy numbers per nucleus may vary among decreases following additions of interspecific propagules could be AMF isolates (38), and the overall high levels of G. intraradices more parsimoniously explained as consequences of more inten- copy numbers, compared to the other two isolates, cannot be un- sive competition for space and carbohydrates (11–14). ambiguously related to high levels of biologically relevant units The observed inoculation effects thus highlight the dynamic such as nuclei or intraradical fungal structures. It is therefore pref- character of both the root colonization process and interactions of erable to avoid direct comparisons of nrDNA copy numbers coexisting AMF observed in earlier studies (41, 43). Nevertheless, among AMF taxa and perform comparisons among experimental an AMF community in responding to inoculation tends to stabi- Downloaded from treatments, such as by using the RII index. lize in most treatments at the end of the cultivation period of 24

The RIIcommunity index clearly showed that G. mosseae was weeks, which indicates its resilience to shifts in propagule densi- competitively the weakest isolate of the synthetic community, ties. while G. intraradices and G. claroideum were more successful com- Inoculation effects on plant performance. The overall posi- petitors, maintaining their abundance when challenged by the tive plant growth response to inoculation into the control back- presence of the other two AMF. This competitive weakness of G. ground showed that all three isolates behaved as mutualists, as mosseae corresponded with the low infectivity of its inocula as would be expected from the vast literature previously demonstrat- determined in the MPN tests. Both features may be related to the ing this (3). The dependence of G. mosseae on inoculum additions different origin and cultivation history of this isolate (23) in com- to improve the plant performance relates to the slower establish- http://aem.asm.org/ parison with G. intraradices and G. claroideum and may reflect its ment of symbiosis with the 20-IP treatment (44, 45). The high lower compatibility with the experimental conditions. 400-IP level of G. intraradices and G. claroideum, in contrast, did Quick development of root colonization by the AMF back- not further improve plant performance compared to the 150-IP ground affirms the generally high inoculation potential of the pre- level, indicating that 150 IP was sufficient to induce maximum cultivated AMF community. Under such conditions, inoculation benefit to the plant. The overall magnitude of the response in the may not be expected to increase root colonization further but control soil, however, and especially the differences between plant instead lead to shifts in the abundances of the community mem- growth in the control and in the AMF background soil should not bers (18, 39). However, the expectation that inoculum additions be overinterpreted in view of other potential microbial factors.

increase the abundance of the inoculated fungus was only con- Soil microbial community and especially rhizobia associated with on November 27, 2018 by guest firmed for the G. mosseae isolate. It is known that the root coloni- legumes such as Medicago may significantly influence plant per- zation of monoinoculated Glomus isolates typically reach a maxi- formance (46). Despite the additions of microbial filtrates, rhizo- mum level over time, depending on plant and fungal isolate bia (and other microbes) were certainly less abundant in the con- identity, as well as environmental conditions. For instance, higher trol soil than in the AMF soil in the beginning of the cultivation, infectivity in soil accelerates the establishment of this plateau level which may explain the generally better performance of plants but does not increase its height (19, 35, 40, 41). Our results dem- growing in AMF soil compared to control soil. onstrated that the G. mosseae isolate was suppressed below this However, this factor does not preclude comparisons among potential maximum by interspecific competition and therefore the inoculation treatments that were dependent upon the AMF could profit in its competitive advantage for host roots from ad- background soil, as the microbial community developed in the ditional propagules. In contrast, the other two isolates may have precultivation step was preserved. The negative growth response achieved their potential maximum abundance within the AMF to inoculation after 6 weeks is interesting and may imply increased community and thus did not respond to intraspecific inoculation competition among AMF following inoculum additions. Bennett by a higher abundance. and Bever (12) raised a question regarding resource allocation in The simple concept of a colonization plateau level, however, AMF between competition and providing benefits to the host fails to explain the observed abundance decreases of G. claroideum plant. The increased competition following inoculation due to and G. intraradices following intraspecific inoculation, especially higher propagule number and disturbance of the preestablished with 400 IP, and these findings point to more complex dynamics propagule balance possibly redirects resources to competitive in- of the root colonization process. The plateau level is a feature of teractions. For example, the competing AMF may have invested microscopically determined root colonization dynamics after more into the formation of structures related to carbon consump- nonvital staining of all fungal structures. In contrast, vital staining tion and space occupation than in structures involved in nutrient and quantification of fungal nuclei based on nrDNA copy num- uptake and transfer to the host. This also supports the hypothesis bers often reveal a peak of fungal vitality after a few weeks of described by Gange and Ayres (47), who proposed a model in cultivation, followed by a decline (19, 22, 35, 42). Assuming accel- which the relation between plant benefit and mycorrhizal density eration of the root colonization process by higher propagule lev- was curvilinear, or rather hump-shaped, where there is an optimal els, the negative abundance responses to intraspecific inoculation range of AMF density for maximum plant benefit and a negative with G. intraradices and G. claroideum may in fact reflect shifts in plant response when AMF density approaches its maximum. colonization dynamics, with an earlier vitality peak and onset of Conclusion. Our results suggest potentially undesirable effects the vitality decline, both occurring before the first harvest after 6 of AMF inoculation in systems where an AMF community is es- weeks of cultivation. Indeed, the G. claroideum isolate displayed tablished. The observed changes in the AMF community and

October 2013 Volume 79 Number 20 aem.asm.org 6513 Janoušková et al. plant performance indicate there may be an optimal level of AMF 19. Thonar C. 2009. Synthetic mycorrhizal communities: establishment and propagule density and composition in soil. Changes in these levels functioning. Ph.D. dissertation. ETH Zürich, Zürich, Switzerland. doi:10 may then lead to increased competition among the root-coloniz- .3929/ethz-a-005927506. 20. Gustafson DJ, Casper BB. 2006. Differential host plant performance as a ing AMF and decrease the AMF community potential to promote function of soil arbuscular mycorrhizal fungal communities: experimen- plant growth. This ecological aspect has been rather neglected in tally manipulating co-occurring Glomus species. Plant Ecol. 183:257–263. inoculation studies and should be further explored. 21. Gryndler M, Sudová R, Püschel D, Rydlová J, Janoušková M, Vosátka M. 2008. Cultivation of high-biomass crops on coal mine spoil banks: can ACKNOWLEDGMENTS microbial inoculation compensate for high doses of organic matter? Bioresour. Technol. 99:6391–6399. This work was supported by the Grant Agency of the Czech Republic, 22. Krak K, Janoušková M, Caklová P, Vosátka M, Scarontorchová H. 2012. number 526/09/0838, and the institutional projects AV0Z60050516 and Intraradical dynamics of two coexisting isolates of the arbuscular mycor- Z5038910 of the Grant Agency of the Academy of Sciences of the Czech rhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR Republic. C. Wagg was supported by a grant from the Swiss National of mitochondrial DNA. Appl. Environ. Microbiol. 78:3630–3637. Science Foundation (number 406840_143097). 23. Enkhtuya B, Rydlová J, Vosatká M. 2000. Effectiveness of indigenous We are grateful to Jana Maršícˇková for excellent technical assistance. and non-indigenous isolates of arbuscular mycorrhizal fungi in soils from Downloaded from degraded ecosystems and man-made habitats. Appl. Soil Ecol. 14:201– REFERENCES 211. 1. van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streit- 24. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. wolf-Engel R, Boller T, Wiemken A, Sanders IR. 1998. Mycorrhizal 1997. The ClustalX windows interface: flexible strategies for multiple se- fungal diversity determines plant biodiversity, ecosystem variability and quence alignment aided by quality analysis tools. Nucleic Acids Res. 24: productivity. Nature 396:69–72. 4876–4882. 2. Wardle DA, Bardgett RD, Klironomos JN, Setala H, van der Putten 25. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment WH, Wall DH. 2004. Ecological linkages between aboveground and be- editor and analysis suite. Nucleic Acids Symp. 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6514 aem.asm.org Applied and Environmental Microbiology AMF Inoculation into a Background Community

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October 2013 Volume 79 Number 20 aem.asm.org 6515 Chapter 8

Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management

Andrea Berruti, Roberto Borriello, Alberto Orgiazzi, Antonio C. Barbera, Erica Lumini and Valeria Bianciotto

Additional information is available at the end of the chapter http://dx.doi.org/10.5772/58231

1. Introduction

Arbuscular Mycorrhizal Fungi (AMF) are a group of obligate biotrophs, to the extent that they must develop a close symbiotic association with the roots of a living host plant in order to grow and complete their life cycle [1]. The term “mycorrhiza” literally derives from the Greek mykes and rhiza, meaning fungus and root, respectively. AMF can symbiotically interact with almost all the plants that live on the Earth. They are found in the roots of about 80-90% of plant species (mainly grasses, agricultural crops and herbs) and exchange benefits with their partners, as is typical of all mutual symbiotic relationships [2]. They represent an interface between plants and soil, growing their mycelia both inside and outside the plant roots. AMF provide the plant with water, soil mineral nutrients (mainly phosphorus and nitrogen) and pathogen protection. In exchange, photosynthetic compounds are transferred to the fungus [3].

Taxonomically, all AMF have been affiliated to a monophyletic group of fungi, i.e. the Glomeromycota phylum [4]. They are considered to be living fossils since there is evidence that their presence on our planet dates back to the Ordovician Period, over 460 million years ago [5]. Investigations on AMF taxonomy began in the nineteenth century with the first description of two species belonging to the genus Glomus [6]. Since that date, many Glomer‐ omycotan species, genus and families have been discovered and characterized by means of traditional approaches based on the phenotypic characteristics (mainly spore morphology). Molecular DNA sequencing-based analyses have recently contributed to a great extent by shedding light on a previously unseen and profound diversity within this phylum [7].

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 160 Biodiversity - The Dynamic Balance of the Planet

Nevertheless, an open debate on the phylogeny of AMF, and in particular concerning some taxonomical groups, is still puzzling scientists [8–10] (Figure 1). Besides a general disagreement about the number of families and genera (Figure 1), what emerges from reference [8] is that Gigasporales are considered to be a separate order from Diversisporales. This is different from what has been reported in the tree on the right side of Figure 1, which was presented in reference [9], and supported by the recent reference [10].

Functionally, AMF form the so-called arbuscular mycorrhizae with plant roots. The most typical AMF structure, which also gives the name to this group of fungi, is the arbuscule (Figure 2). This structure, whose shape recalls that of a small shrub, forms inside the root cortical cells by branching in several very thin hyphae. In this way, the surface area, where the nutritional exchanges between the plant and fungus take place, is maximized. Fungal hyphae that grow between root cortical cells are able to produce other AMF structures, such as intercellular hyphae and vesicles (Figure 2). All these structures that grow inside the plant roots represent the intraradical phase of the fungus. Hyphae also grow outside the plant roots, and generate a network that extends over long distances and explores the soil beyond the nutrient depletion zone that normally characterizes the area surrounding the roots. At the end of the AMF life cycle, or in response to particular environmental conditions, spores (Figure 2) of variable size (up to 400 µm), depending on the species, are produced in the roots and/or in the soil. These, along with external explorative and running hyphae, represent the extraradical phase of the fungus. The synergic action of the intra-and extraradical phases is responsible for the ecological significance of the AMF, a soil-root-living key group of organisms [3].

1.1. The ecological roles of AMF

Arbuscular mycorrhizal fungi have a high relevance in many ecosystem processes. Since they can be found in many different plant species, they can provide their favorable services to almost all terrestrial ecosystems, from grasslands to forests, deserts and agroecosystems [11]. AMF can play several roles in such environments. The most agriculturally significant and frequently investigated one, from both the ecological and physiological points of view [12], is their positive effect on plant nutrition and, consequently, on plant fitness. In particular, they play a pivotal role in helping the plant uptake phosphorus from the soil [13]. Without AMF, it is rather difficult for the plant to absorb this macroelement from the soil, since it is mainly available in its insoluble organic or inorganic form. Besides phosphorus, AMF can also translocate water and other mineral nutrients (in particular nitrogen) from the soil to the plant. These nutritional exchanges are bidirectional. As a consequence, particularly efficient symbiotic associations have been demonstrated to stabilize through unknown mechanisms, with the plant selecting the most cooperative fungal partners and vice versa [14]. The AMF-inducible recovery of plant nutritional deficiency can inevitably lead to an improvement in plant growth, with a potential positive impact on productivity. Needless to say, AMF have attracted a great deal of interest from the agricultural world over the years [15].

AMF are also responsible for other services that favour the plants they colonize: (a) they positively affect plant tolerance towards both biotic (e.g., pathogens) and abiotic stresses (i.e., drought and soil salinity) by acting on several physiological processes, such as the production Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 161 http://dx.doi.org/10.5772/58231

Figure 1. A schematic representation of two recently published and partly controversial phylogenetic trees of the Glomeromycota phylum (reference [8] for the tree on the left side and [9] for the tree on the right side). The one pub‐ lished in reference [8] was based on molecular (SSU, ITS, partial LSU rDNA, and partial β-tubuline gene) and morpho‐ logical analyses (spore wall structures, structures of the spore bases and subtending hyphae, germination, and germination shield structures). The tree published in reference [9] was based on concatenated SSU rDNA consensus sequences (ca 1.8 kb). of antioxidants, the increment of osmolyte production or the improvement of abscisic acid regulation [16,17], and the enhancement of plant tolerance to heavy metals [18]; (b) they help plants become established in harsh/degraded ecosystems, such as desert areas and mine spoils [19]; (c) they increase the power of phytoremediation (the removal of pollutants from the soil by plants) by allowing their host to explore and depollute a larger volume of soil [20,21]. Another crucial ecological role played by AMF is their capacity to directly influence the diversity and composition of the aboveground plant community. Several studies have 162 Biodiversity - The Dynamic Balance of the Planet

Figure 2. Extraradical and intraradical phases of AMF growth. The spore (Figure E, a Scutellospora sp. spore stained with Melzer’s reagent and squeezed) germinates in the bulk soil and approaches the root of a host plant. The fungus penetrates through a hyphopodium (Figure A, stained with 0.1% cotton blue encountered in Camellia japonica L. roots) and develops intracellular coils, extracellular vesicles and intracellular arbuscules (Figures B, C, D) within cortical parenchyma, without entering the central cylinder where the vessels are.

confirmed that plant species richness can be altered not only by climatic and edaphic factors, but also by soil microbial assemblages [22–24]. The underlying mechanism is not completely understood, but could be related to the promotion of seedling establishment of secondary plant species [25]. Nevertheless, on some occasions, AMF can also negatively affect the diversity and growth of plants, which is particularly significant for the management of weeds [26]. Last but not least, AMF play a critical role in soil aggregation, thanks to their thick extraradical hyphal network, which envelops and keeps the soil particles compact. It has been suggested that glycoproteins (glomalin and glomalin related proteins) secreted by AMF into the soil could exert a key role in this process [27,28]. These proteins are exuded in great quantities into the soil, and could have implications on carbon sequestration. This potential capability of AMF is

likely to contribute to a great extent to the soil ecosystem carbon dioxide (CO2) sequestration Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 163 http://dx.doi.org/10.5772/58231 process. This aspect has led to the recognition of the importance of this group of organisms in processes related to climate change mitigation [29].

All the services offered by arbuscular mycorrhizal fungi confirm the need to study and describe all their features, including their biology, ecology, taxonomy, phylogeny and biodiversity. Over the years, several techniques have been developed to reach this goal: a brief history is reported in section 1.2.

1.2. Methods used in the study of AMF

This group of organisms has a constraining characteristic that makes their study very complex: as obligate symbionts, they cannot be cultivated in vitro, away from their host plant. The development of an artificial system that is capable of going beyond this barrier dates back to the 1980s, when in vitro transformed carrot roots were successfully colonized by AMF species [30]. Thanks to this method, the study of arbuscular mycorrhizae became easier and many researches on both physiology and genetics became possible [31,32]. Over the last two decades, many molecular and physiological mechanisms involved in the symbiotic process between plants and AMF have been discovered and described, thanks to the increasing innovations and opportunities offered by molecular biology. For example, it is now known how the infectious process of AMF arises, and many of the involved genes have been identified successfully [33].

Molecular biology has also revolutionized the analysis of the biodiversity of AMF, making it easier and more accurate to characterize the AMF community composition of large quantities of samples from many different ecosystems, from prairies to savannas, and from grasslands to forests (Table 1). The first studies on the diversity and distribution of AMF were mainly focused on the identification of the species that colonize the roots of a given plant in a given environment [34]. This was mainly due to the deficiency in the available investigation techniques, as they were primarily based on spore surveys and intraradical fungal structure morphological identification by means of microscopy. Such morphological identification surveys were time consuming and often lacked accuracy, since many species were easily confused with others. The situation changed radically when the use of DNA-based techniques became common, and the extraction of DNA from plant tissue was reduced to a few relatively easy steps that could be reproduced in any laboratory [35,36]. The load bearing principle is simple: by sequencing a specific DNA region, it is possible to univocally identify the corre‐ sponding AMF [37]. So far, the most used DNA target regions for AMF identification are located on the ribosomal genes (Small and Large ribosomal Subunits – SSU and LSU, respec‐ tively – and the Internal Transcribed Spacers – ITS1 and ITS2), as they show a rate of variability that is sufficient to discriminate between AMF species/isolates [9]. All this has led to the current era of molecular identification of AMF species [10]. Next-Generation Sequencing (NGS) tools represent a further step forward for biodiversity surveys of all organisms [38], including AMF. Over the last few years, the number of NGS-based AMF biodiversity studies has increased, while the spectrum of the target environments has broadened [39]. Furthermore, new primer pair sets for the specific amplification of AMF DNA sequences, capable of providing higher accuracy and a comprehensive coverage of the whole Glomeromycota phylum, have been 164 Biodiversity - The Dynamic Balance of the Planet

developed [40]. Nowadays, AMF assemblages are no longer studied only in plant roots, but also in the bulk soil [41–43]. The main result obtained from the application of NGS to the study of AMF biodiversity has been the discovery of an unpredictable diversity within the Glomer‐ omycota phylum [39]. However, this series of innovative molecular tools has introduced a new issue, that is, the continuously increasing number of unidentified AMF DNA sequences from environmental samples with no correspondence whatsoever to sequences of known species [44]. This has naturally made scientists aware of the fact that the number of AMF species could be larger than expected. However, it is not reliable to have new species described on just the basis of short DNA sequences obtained by means of NGS tools. Instead, for each new suggested taxon, a series of steps needs to be followed to characterize the morphotype, the functional traits, and the ecological role offered when present in combination with other organisms in a given environment. Therefore, NGS tools cannot be considered as complete replacements of the traditional methods of identification and description of new species. The combined approach is still necessary to shed light on such a key group of organisms and to make them available for agricultural application and, more in general, for other practices useful for the wellbeing of humankind [45].

4. Target 5. Studied 7. AMF 1. Reference 2. Year 3. Method 6.Ecosystem 8. OTUs region compartment sequences

Tropical, subtropical, temperate and boreal forests, subtropical and temperate grasslands, tropical Clon- [39] 2013 SSU Plant root and subtropical 2353/22391 204 seq/NGS deserts and shrublands, and polar tundras (Africa, Asia, Oceania, Europe, North and South America)

[46] 2013 NGS SSU Soil Prairie (Cananda) 1335521 120

Temperate forest [47] 2013 NGS SSU Plant root and Soil 35738 76 (Estonia)

Mediterranean semi- [48] 2013 Clon-seq SSU Plant root 467 30 arid soils (Spain)

[49] 2013 Clon-seq SSU Soil and plant root Prairie (USA) 232 13

[43] 2012 NGS SSU Soil Forest (Estonia) 13320 37

[50] 2012 NGS SSU Soil Arable field (China) 59611 70 Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 165 http://dx.doi.org/10.5772/58231

4. Target 5. Studied 7. AMF 1. Reference 2. Year 3. Method 6.Ecosystem 8. OTUs region compartment sequences

Prairie - Chernozem [51] 2012 NGS SSU Soil 7086 33 (Cananda)

[52] 2012 NGS LSU Plant root Grassland (Denmark) 82511 32

[42] 2012 Clon-seq SSU/LSU Soil and plant root Arable field (Italy) 427/364 20/23a

Alpine meadow [53] 2012 Clon-seq SSU Plant root 4452 38 ecosystem (China)

Broadleaf, mixed broadleaf and [54] 2011 NGS SSU Plant root coniferous forests, 65001 73 botanical gardens, greenhouse

Grassland, wood and [55] 2011 NGS SSU Plant root 108245 70 heath (UK)

Hardwood forest [56] 2011 Clon-seq SSU Plant root 1598 17 (USA)

Mediterranean soils [41] 2010 NGS SSU Soil 2815 19/80a (Italy)

[57] 2010 Clon-seq SSU Soil and plant root Vineyard (Italy) 681 37

[58] 2009 Clon-seq SSU Plant root Woodland (UK) 617 33/37b

Mediterranean semi- [59] 2009 Clon-seq SSU Plant root 1443 21 arid soils (Spain)

Boreal forest [60] 2009 NGS SSU Plant root 111580 47 (Estonia)

[61] 2008 Clon-seq LSU Soil and plant root Arable field (Italy) 183 8

Boreal forest [62] 2008 Clon-seq SSU Plant root 911 26/27c (Estonia)

[63] 2008 Clon-seq SSU Plant root Arable field (Mexico) 213 16

Serpentine soils [64] 2008 Clon-seq SSU Plant root 1249 19 (USA)

Arable field [65] 2008 Clon-seq SSU Plant root 115 8 (Sweden)

Soil, plant root and [66] 2007 Clon-seq ITS Meadow (Germany) 180 >18 spores

Liverworts (World- [67] 2007 Clon-seq SSU Rhizoids 150 10 wide) 166 Biodiversity - The Dynamic Balance of the Planet

4. Target 5. Studied 7. AMF 1. Reference 2. Year 3. Method 6.Ecosystem 8. OTUs region compartment sequences

[68] 2007 Clon-seq LSU Soil and plant root Arable field (France) 246 12

[69] 2007 Clon-seq SSU Plant root Grassland (Sweden) 185 19

Volcanic desert [70] 2007 Clon-seq ITS Plant root 205 11 (Japan)

[71] 2006 Clon-seq SSU Plant root Polluted soils (Italy) 115 12

Warm-temperate [72] 2005 Clon-seq SSU Plant root deciduous forest 394 5 (Japan)

[73] 2004 Clon-seq SSU Plant root Wetland (Germany) 546 35

[74] 2004 Clon-seq LSU Plant root Grassland (Denmark) 158 11

[75] 2004 Clon-seq ITS Plant root Pasture (UK) 30 10

[76] 2004 Clon-seq SSU Plant root Grassland (Japan) 200 8

[77] 2004 Clon-seq SSU Plant root Grassland (UK) 606 9

Afromontane forests [78] 2003 Clon-seq ITS Plant root 92 20 (Ethiopia)

Boreal forest [79] 2003 Clon-seq SSU Plant root 16 6 (Estonia)

Seminatural [80] 2002 Clon-seq SSU Plant root 88 24 grassland (UK)

[81] 2002 Clon-seq SSU Plant root Woodland (UK) 232 13

Tropical forest [82] 2002 Clon-seq SSU Plant root 1536 18/23d (Republic of Panama)

Tropical forest [83] 2002 Clon-seq SSU Plant root 558 18 (Republic of Panama)

[84] 2001 Clon-seq SSU Plant root Arable field (UK) 303 8

Seminatural [36] 1999 Clon-seq SSU Plant root 141 6/8e woodland (UK)

[85] 1998 Clon-seq SSU Plant root Woodland (UK) 253 6/10b

a-taxa obtained with different primer sets; b: taxa obtained at different study sites; c-taxa obtained from forest ecosystems of different ages and management intensities; d-taxa obtained from roots of different plant species; e-taxa obtained at different sampling times.

Table 1. The table shows an overview of DNA-based studies on the diversity of Arbuscular Micorrhizal (AM) fungal communities. For each study, the following are reported in sequence: 1. Reference, 2. Year of publication, 3. Used method (Clon-seq=cloning and sequencing; NGS=next generation sequencing), 4. Studied DNA region (SSU=Small Subunit; LSU=Large Subunit, ITS=Internal Transcribed Spacer), 5. Compartment from which the DNA was analyzed, 6. Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 167 http://dx.doi.org/10.5772/58231

Ecosystem from which the samples were collected, 7. Number of DNA sequences and 8. OTUs (Operational Taxonomic Units) from arbuscular mycorrhizal fungi.

2. The impact of humans on AMF biodiversity

Most human activities have an arguable impact on the physical and biological aspects of soil. As mentioned before, AMF are among the most widespread soil microorganisms, and each human activity that has an impact on soil, such as agricultural practices, therefore has a side effect on them. These practices, alone or in combination, exert an enormous selective pressure on AMF that shapes their community structure and evolution by modifying several of their biological features, such as sporulation strategy, resource allocation and spatial distribution [86]. As in natural ecosystems, AMF are also present and active in agricultural ecosystems, where they colonize several major arable crops (sorghum, maize, wheat and rice). Many studies have indicated that AMF diversity, effectiveness, abundance and biodiversity decline in agroecosystems subjected to high input practices [41,42]. Modern intensive farming practices that implement deep and frequent tillage, high input inorganic fertilization and pesticide use are evidently a particular threat to AMF. This is surely a drawback for agriculture, since the more AMF biodiversity losses, the fewer AMF functional traits the host plant can benefit from. On the other hand, the activity and diversity of AMF, following conversion from conventional to organic farming, have not yet been investigated thoroughly. However, the available data seem to indicate that AMF respond positively to the transition to organic farming through a progressive enhancement of their activity [87]. Even though it is difficult to dis‐ criminate between the effects that different agricultural treatments exert on AMF communities, they are here considered separately, and their role in shaping AMF communities will be analyzed.

2.1. Tillage: A conventional practice detrimental to AMF

One of the most ancient and representative agricultural techniques is tillage. Tillage has played a crucial role in the evolution and technological development of agriculture, particularly for food production. The benefits produced by tillage include a better conservation of water and soil fertility, the abatement of weeds and the preparation of a suitable seedbed. To fulfill these tasks, the undisturbed soil is mechanically manipulated in an effort to modify the physical characteristic of the soil and eliminate weeds. The physical, chemical and biological effects of tillage on the soil can be both beneficial and negative, depending on the methods that are used. The inappropriate use of tillage techniques can therefore have a dramatic impact on the soil structure and on soil microorganism community assemblage. It is possible to identify different tilling levels, ranging from a very low impact, “No-tillage”, to a high impact, “conventional tillage”. A continuum of intermediate conditions lies in between these two extreme situations, e.g. varying frequency and intensity of the plowing. The mechanical soil disturbance experienced by AMF in tilled agricultural soils has no equivalent in natural ecosystems. This is why tillage has been widely recognized to be one of the principal causes of the modification of the AMF communities that colonize plant roots in 168 Biodiversity - The Dynamic Balance of the Planet

agricultural fields [88]. Mycorrhizal diversity, at a family level [88], and the timing of root colonization [89] can be affected negatively. As a consequence, the effectiveness of AMF [90] is likely to be reduced. Periodically repeated mechanical soil disturbance destroys the extra‐ radical mycelial network formed by AMF. This very complex underground structure can reach lengths of up to some tens of meters in one gram of soil [91], and represents a soil “highway” for nutrient transport. For this reason, it is often claimed to be closely correlated to biodiversity, biomass production and the functioning of plant communities [22,25,92].

An ecological shift in AMF communities is particularly noticeable when frequently and infrequently tilled agroecosystems are compared [42,63,88,93]. This is probably due to the different tolerance to hyphal disruption among the different AMF species [94,95]. Although AMF species can colonize plants from spores, this process often requires a certain amount of time. Faster root colonization can be reached in the presence of a viable and well-structured underground mycelial network that facilitates AMF proliferation and speeds up plant root penetration [96]. On the other hand, AMF species differ greatly in their capacity to restart colonization from fragmented mycelium or root fragments [97]. Intense tillage could be a factor that favors those AMF species that are more able to proliferate from fragmented hyphae or root fragment [98], and could therefore determine a shift in AMF community assemblages. A clear example of this is the large presence of Glomeraceae species found in tilled soil all over the world [99]. AMF species belonging to this group are able to randomly connect hyphae in close proximity after disruption, a condition that can easily be found in disturbed soil. This allows these species to proliferate more easily and to rapidly become dominant over slow- growing AMF. The members of the Gigasporaceae family, for example, use spores as the main source of root colonization, but do not regrow from hyphal fragments [97].

2.2. Fertilization

Another agricultural practice that has major ecological fall-outs is chemical fertilization. This practice is often claimed to be fundamental in improving the growth performance of plants, but it is sometimes abused. In addition to the environmental drift and the possible pollution of underground water reservoirs, the presence in the soil of high levels of fertilizer dramatically alters the interaction between plants and microbial communities. The central role of arbuscular mycorrhizae in plant nutrition makes them very susceptible to changes in soil nutrient availability. Generally, in a nutrient-rich environment, a plant can directly uptake enough nutrient from the soil, without the “catering” service provided by the AMF partners. As a result, the dependency of plants on their AMF partners gradually diminishes, and AMF community richness and diversity decline [42,53,100,101]. It is thought that fertilization can alter the performance of this symbiosis, making microbial partners costly, and even parasitic [102]. It has been hypothesized that the enrichment of soil resources, due to high input fertilization, could lead to a reduction in plant allocation to roots and mycorrhizas [103], and an accumulation of nutrient resources in epigeous plant sinks [104]. A reduction in host plant resource allocation to the fungal partners can therefore result in a decrease in AMF root colonization [105], and an increase in fungal competition for limited C resources. Moreover, this reduction in host nutrient availability is thought to shift the competitive balance between Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 169 http://dx.doi.org/10.5772/58231 microbes, favoring more aggressive, antagonistic microbial genotypes [106–108]. This change in competitive balance can alter the evolution of the functional traits of AMF by reprogram‐ ming AMF to reduce their allocation to structures devoted to nutrient exchange (arbuscules and coils), and increase their allocation to internal storage and growth structures (vesicles and intraradical hyphae) [103,109,110]. This is likely to result in an incremented presence of highly competitive AMF which, on the other hand, will be less beneficial to the host crop [111].

Particular AMF taxa have been found to be more sensitive than others to specific fertilization conditions [42,50,53,65,93,112]. This is probably due to the different taxon-related ability of the AMF taxa to manage nutrient absorption. For instance, Acaulospora species have been dem‐ onstrated to be very effective in P uptake, and in the transfer to the host plant, compared to Glomeraceae species [113]. In line with these findings, Acaulosporaceae species have been considered to decrease to a great extent under high input P fertilization [50]. The same thing has been observed for Gigasporaceae in N-enriched soils [50,103]. On the other hand, Glom‐ eraceae species, such as Rhizophagus intraradices, are able to cope well with nutrient rich environments [50,53].

2.3. Crop rotation

The choice of crop and rotation made by the farmer has a crucial impact on AMF communities. Even though AMF are commonly recognized as generalist symbionts that show the ability to interact with different plant species, some plant-fungus combinations can perform better than others. The choice of the partner is not univocal, but is believed to be driven by a reciprocal reward mechanism between the two symbionts involved [14]. This means that both the plant and the AMF communities can exert an important role in modifying the community compo‐ sition of the partner [22,23]. Thus, different cultivation practices that involve a variation in plant diversity, such as monoculture, fallow and crop rotation, could show different and profound effects on AMF community assemblages.

Monoculture can be highly deleterious for AMF communities, and result in a significant reduction in mycorrhizal root colonization [114] and mycorrhizal diversity [115,116]. The effect of continuous monocropping, especially when crops that are not highly dependent on AMF- mediated nutrition (e.g. wheat) are used, favors the selection and proliferation of less cooper‐ ative and more aggressive fungal symbionts. These are likely to enact similar behavior to parasitism [102,106]. In addition, intensive tillage treatments, which are necessary in the case of monoculture practices, can overly disperse fungal propagules, thus allowing fewer AMF isolates to dominate the community profile. The dominion of AMF species with a poor mutualistic attitude could be toned down by alternating the cultivation of plant species that are less dependent on AMF with ‘break crops’, such as Brassica [117] or legumes [118]. The former is a non-mycorrhizal crop that can therefore act as an inhibitor of the dominant AMF species proliferation. The latter represent the opposite approach, since legumes are AMF- dependent crops that favor the overall propagation of AMF communities. This is the funda‐ mental principle of crop rotation, a practice that can exert a control function that prevents particular AMF from dominating the soil matrix. Hence, crop rotation has the potential of driving AMF communities to be less parasitic [86]. It has been experimentally demonstrated 170 Biodiversity - The Dynamic Balance of the Planet

that crop rotation promotes higher AMF diversity [115,119], and can reshape AMF commun‐ ities derived from agricultural fields to be more diverse and similar to the ones detected in natural ecosystems [87].

3. AMF biodiversity restoration

Agricultural fields, degraded lands and the so-called “third landscapes” are all soil environ‐ ments in which humans have had an impact on the ecological balances, by unchaining a series of inevitable ecosystem alterations. Therefore, the restoration of such balances should be a necessity. Owing to their role in the promotion of plant health, soil nutrition improvement and soil aggregate stability, AMF are primary biotic soil components that, when missing or impoverished, can lead to a less efficient ecosystem functioning. The presence of a high degree of AMF biodiversity is in fact typical of natural ecosystems and indicates good soil quality [120]. Consequently, a process that aims at the re-establishment of the natural level of AMF richness is a pivotal step towards the restoration of the ecological balances. As previously mentioned, the cultivation practices adopted for major crops include anthropic inputs that can impact AMF occurrence and/or diversity. Of these, the use of fertilizers and pesticides also has an adverse impact on production costs, and should be reconsidered due to the heightened social concern about the corresponding environmental drift [121]. As a consequence, the need to benefit from AMF as a biofertilizer, with a view to sustainable agriculture, is becoming increasingly urgent. An appropriate management of these symbiotic fungi would lead to a great reduction in chemical fertilizer and pesticide inputs, a key target for growers facing a crisis, and having to deal with a more environmentally aware clientele. Two main strategies are possible to achieve this goal: the direct re-introduction of an AMF pool (referred to as “inoculum”) into the target soil, or the selective management of the target ecosystem. These strategies can be selectively adopted when a population of AMF propagules of low effectivity is present, or when the indigenous AMF are absent or very low. This means that the AMF restoration process is suitable for different purposes, e.g. greenhouse and open-field cultiva‐ tion, and even in helping the rehabilitation of degraded lands.

3.1. AMF inoculation and the role of enterprises The re-introduction of AMF into soils that are impoverished in belowground biodiversity is a complex strategy, but it can be very rewarding. Unfortunately, the production of AMF inoculum on a large-scale is very difficult using the techniques currently available. The main obstacle to the production of an AMF inoculum lies in their peculiar symbiotic behaviour, the AMF compulsorily requiring a host plant for growth. This means that AMF are propagated through cultivation with the host plant, and this usually requires time-demanding protocols and cumbersome infrastructures. The maintenance of AMF reference collections requires methodologies that are rather different from those used for other microbial collections and inoculum production. Unlike non-obligate symbionts, the production of AMF inoculum requires the control and optimization of both host growth and fungal development. Thus, these propagation techniques involve high costs that are not apparently competitive with fertiliza‐ Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 171 http://dx.doi.org/10.5772/58231 tion-related costs. The impossibility of rapidly assessing AMF colonization on the host plant, together with the complexity of AMF species identification, also contribute to the pitfalls of inoculum agricultural usability. Moreover, the management of the high amount of inoculum necessary for extensive use is very challenging. It has been suggested that AMF is more suitable for plant production systems that involve a transplant stage, as inoculation is carried out more easily, and smaller quantities of inoculum are needed. At a first glance, establishing an open- field, large-scale inoculation treatment would seem technically impractical and economically prohibitive. However, once AMF biodiversity has been restored, AMF-friendly practices, such as fall cover cropping [122], can be put in place in order to help the AMF persist. If no detri‐ mental agricultural practices are carried out, the biodiverse mycelial network will remain unaltered and infective in the future. For example, in revegetation schemes, it would be totally impractical to restore an entire degraded land, which often appears as a highly extended surface, through inoculation. A particular approach must be considered when it is necessary to face these situations. First, the ability of specific cover crop mixtures and even target indigenous plant species to elevate the native AMF inoculum has to be taken into account as a potentially successful selective management tool to aid the recovery of desertified ecosystems [123]. However, since ecosystem functioning is supported by a close liaison between the aboveground plant diversity and belowground AMF diversity [22], the excessive loss of AMF propagules in degraded ecosystems could, in some cases, preclude either natural or artificial revegetation. For this reason, an inoculation step may also be needed. Although it would be too laborious and expensive to re-introduce AMF and cover plants into entire lands, a smaller- scale approach should be adopted. Taking inspiration from the idea of creating the so-called “fertility islands” [124], only small patches of cover plants could be inoculated with AMF. This could lead, in time, but with reduced costs, to the re-establishment of a mycelial network that would also be able to allow native plant species to quickly recover the nutrient impoverished land.

Hence, AMF restoration would only represent an initial cost and, if soil AMF persistence is favoured, this cost could be subjected to amortization over the years. This makes the applica‐ tion of AMF particularly attractive since, as already demonstrated [125,126], it could provide considerable savings for growers and for degraded land recovery projects, in comparison to conventional fertilization. It is important that the end-users cultivate a portion of their crop without inoculum in order to assess the cost-effectiveness and the beneficial effects on plant fitness due to AMF inoculation [127]. Growers are starting to understand the significance of sustainable agricultural systems, and of reducing phosphorus inputs using AMF inocula, especially in the case of high value crops, such as potted ornamental plants. These crops can easily be regarded as the result of organic crop farming, and be sold at a premium price to an eco-friendly orientated consumer class. However, the absence of solid inoculation practices still represents a problem, and applied research should therefore be focused on defining the best inoculum formulation strategies [128] and imparting know-how to the growers.

Since large-scale AMF production is impractical for growers, the significance of AMF has not been ignored by the commercial sector, and many AMF-based inocula are nowadays available for sale. AMF inoculum production began in the 1980s and flourished in the 1990s. Nowadays, 172 Biodiversity - The Dynamic Balance of the Planet

several companies produce and sell AMF inocula. In recent years, these products have come under increasing scrutiny by scientists and end-users. Most manufacturers advertise their products by pointing out their suitability for a wide range of plants and environmental conditions. Unfortunately, their promises made about these products and the results seen are too often worlds apart. This has led to radical generalisations, both positive and negative, about the efficacy of the currently available products. The problem is that success, in terms of root colonization and plant response, is unpredictable since no plant does best with the same AMF mix [129]. In terms of fungal content, the manufacturer’s tendency is to introduce a more or less biodiverse mix of AMF. Some companies have chosen the approach of single formulations, while others produce a range of differently shaped products for their target end-users. Glomeraceae species are usually used, but also Gigasporaceae, Scutellosporaceae and Acau‐ losporaceae families are gradually being introduced to commercial inoculum production. These few used species can be routinely propagated for spore applications, are found in association with a large variety of host plants and are geographically distributed all over the world.

Great problems arise in formulating the inoculum product in its most suitable state for the market. In the coming years, it is likely that greater regulation and controls will be introduced concerning the production and selling of AMF inocula. In Europe, the regulation of these products varies from country to country, with some having very strict regulations, while others are less demanding. In North America, Canada, for instance, considers AMF inocula to be only supplements and not fertilizers. In the USA, registration may fall either to the fertilizer or the pesticide sectors, depending on the supposed action of the formulated AMF inoculum. However, in most countries, AMF are no longer considered dangerous for human or animal health, and no infectivity or toxicity tests are therefore necessary. Normally, an application for registration has to be filled in and a series of meticulous information needs to be attached to the registration request. These data should also be reported on the inoculum label, and should include the list of all the ingredients and their concentrations, a detailed taxonomic description of the AMF, the isolate’s history, the geographic origin and distribution, some literature on the beneficial effects of the isolate, a list of possible contaminants, an official safety data sheet, information about the producer, the number of viable AMF propagules or the percentage of colonization expected on reference plants after a known quantity is inoculated, the list of recommended plant hosts, the suggested soil conditions for inoculum effectiveness, the recommended application method/dosage, the suggested storage conditions, the expiration date and information on the manufacturing processes. Other information regarding previous tests performed with different soil, and which confirms the climatic conditions and the beneficial effect of the inoculum should also be added in order to highlight the reliability of the product and to help direct the consumer. Preventing over-regulation will be crucial in assisting the development of SMEs (Small and Medium Enterprises), and in helping refresh the market with this eco-friendly biotechnological tool.

In order to allow the AMF inoculum market to develop, scientists should define a series of 'best practices' that could be adopted by these SMEs to solve serious issues related to their product quality. One of these issues arises from the need to control the biological composition Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 173 http://dx.doi.org/10.5772/58231 of the product, especially for the possible presence of pathogens, but above all to assess its quality in terms of AMF composition. Being obligate symbionts, AMF are non-axenically culturable, while only a few can be monoxenically cultured. Therefore, an inoculum is produced above all using a containerized-culture, either in greenhouses, growth chambers, or in fields, and, as a result, cannot be completely free from external microorganisms. There is increasing awareness of the risk of pathogens, and many concerned producers are even making use of agrochemicals in an attempt to avoid contamination of their product. Others have instead decided not to include host root residues in their formulation, in order to avoid pathogen carry-over. Alternatively, surface sterilization of the incorporated colonized roots can be introduced without affecting the viability of the AMF propagules [130]. As far as quality control in terms of AMF composition is concerned, it is essential to verify whether the product effectively has the potential described on the label. With AMF, in order to confirm the fungal identity, such an assessment can be done through morphological identification of the spores [131,132]. Unfortunately, this technique requires a great deal of labor and there are very few experts in the world that are able to conduct a reliable identification solely on the basis of spore morphology [133]. Quick and user-friendly molecular techniques have been developed to detect AMF strains from complex matrices, such as soil [41,42] and AMF inocula [129,134]. The discrimination of AMF, on the basis of these techniques, relies almost completely on the sequencing of the ribosomal genes, the genetic region on which the AMF phylogenesis was constructed (4), and is still under debate [8–10]. Molecular techniques also allow the inoculated isolates to be reliably traced inside the host plant and their persistence in the soil to be established [135]. The use of Realtime qPCR and specific primers appears to be a very prom‐ ising tool for the tracing of AMF isolates and their quantification in the host roots after application [136]. A recent study has even used laser microdissection to qualitatively monitor the arbuscule formation in Camellia japonica L., after inoculation with a highly biodiverse AMF inoculum [134]. Such a quality control is very important to exclude poor quality or defective AMF inocula from the market.

3.2. Key steps and current techniques for inoculum production

The actual inoculum propagation and formulation process entails a series of key steps that are crucial for the good quality of the final product. The most determining aspect of inoculum formulation is the choice of the AMF content. As mentioned before, the tendency is to introduce a mix of several AMF into commercial inocula. The most scientifically investigated AMF isolate, i.e. Rhizophagus irregularis DAOM197198 [137], is also one of the most frequently used for commercial inoculum formulation. This species is a very generalist symbiont that can colonize a large variety of host plants, survive long-term storage, is geographically distributed all over the world and, last but not least, adapts well to both in vivo and in vitro propagation. These characteristics make this isolate of R. irregularis suitable to be a premium component of commercial inocula. As previously mentioned, several other AMF that mainly belong to Glomeraceae species, but also to Gigasporaceae, Scutellosporaceae, and Acaulosporaceae families, are gradually being introduced into commercial inoculum production. It is important to notice that AMF are sometimes marketed as consortia that contain ectomycorrhizal fungi, saprophytic fungi and plant growth-promoting rhizobacteria (PGPR), in order to increase the 174 Biodiversity - The Dynamic Balance of the Planet

product potential for plant protection and production. The proper choice of the inoculum AMF content is unfortunately constrained by a lack of knowledge on the specificity of the relation‐ ships between a specific AMF strain and a particular crop, and on the compatibility and competition of the AMF strains for niches in the soil environment [128]. When AMF are examined as a community, there is abundant evidence that fungal growth rates can be host- and niche-specific. In reference [60], it has been suggested that partner specificity in AM symbiosis may occur at an ecological group level of both the plant and fungal partners. In [14], it has been demonstrated how reciprocal "rewards" stabilize cooperation between the host- plant and the fungus, thereby enforcing the best symbiotic combinations. Thus, the best way of finding the most cooperative and specific AMF isolates for the formulation of more targeted inocula is to directly screen what nature offers, by fathoming out the naturally occurring symbiotic combination set. For example, some AMF species are commonly recognized to be more stress tolerant than others, and are usually found in stressed and polluted soils [18,138]. Native AMF from areas affected by osmotic stresses can potentially cope with salt stress in a more efficient way than other fungi [139]. Thus, it is preferable to take this into account when “tuning” an inoculum to a particular kind of degraded/stressed soil and in order to avoid failure of the revegetation process [140,141]. Optimal benefits will only be obtained from inoculation after a careful selection of the favorable host/niche/fungus combinations. For this reason, natural or semi-natural ecosystems, in which the desired host plant is well established, represent a valid source of naturally selected AMF. However, this highly selective inoculum formulation requires time and hard work. An intriguing approach would be to formulate a series of highly biodiverse inocula, including several AMF species/strains of different geo‐ graphical/environmental origin, which would be capable of offering benefits to multiple host plants under different environmental conditions, thus making researchers switch from looking for a superstrain to formulating a superinoculum.

AMF can use a number of different types of propagules to colonize new roots with different degrees of efficiency [142]. These are components of the extraradical and intraradical phase of AMF. The extraradical phase comprises spores and a mycelium that forms the hyphal network. Several fungal structures, inside both living and dead root fragments, can represent a source of inoculum [143]. Vesicles, in particular, have been shown to be very infective [97]. Consid‐ ering that a number of different propagule types exist, it is of primary importance to determine the most eligible and user-friendly to be adopted as inoculum sources. Unfortunately, this is more complex than may be expected, since different AMF taxonomical ranks differ in their ability to propagate from a given propagule. As already mentioned, for instance, it seems that propagation through mycelial fragmentation may be more important for species of the Glomeraceae family, whereas spore germination may be the preferential type of propagation for species in other families (e.g. Gigasporaceae). In reference [144], the authors tested the establishment of a biodiverse community of AMF in a pot culture using different sources of inoculum from the field. They found that spores were successful in establishing most species of Acaulosporaceae, Gigasporaceae and Scutellosporaceae, whereas Glomeraceae species were only dominant when root fragments or soil cores were used. It is important to consider that these different propagation strategies can also reflect on the potential agricultural use of a particular AMF inoculum. Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 175 http://dx.doi.org/10.5772/58231

Once the AMF content has been selected, pure monospecific cultures are normally obtained from a single spore, or a small piece of colonized root fragment, or mycelium collected directly from field plants, or obtained from AMF collection cultures. The AMF propagule spreads and colonizes the root apparatus of the host plant, and the subsequent pot-culture generations lead to the production of high quantities of AMF inoculum. Several organizations throughout the world have research culture collections (The International Culture Collection of VA Mycor‐ rhizal Fungi, INVAM; The Banque Européenne des Glomales, BEG; The Canadian National Mycological Herbarium, DAOM; The Canadian Collection of Fungal Cultures, CCFC; The non- profit Biological Resource Center ATCC; The Glomeromycota In Vitro Collection, GINCO; NIAS, National Institute of Agribiological Science) and provide users with reliable AMF propagules to start propagation. Moreover, detailed information on species origin and distribution, spore morphology, and molecular biology and biochemistry are often provided by these organizations. The common purpose of these available AMF collections is to provide a stock source of pure and reliable material for fundamental and applied research use.

A pivotal step during AMF inoculum propagation is the choice of an adequate host plant. The criteria required for the host plant are its high mycorrhizal dependency and potential, i.e. its capacity of being highly colonized by a high number of AMF species, and its inclination to promote growth and sporulation, its suitability to grow under growth chamber or greenhouse conditions and its production of an extensive root system with a high number of fine feeder roots in a short time. A series of plants are commonly recognized as actual AMF “trap” plants, due to their mycorrhizal dependency and lack of specificity, and they are routinely used as host plants during propagation. These include clover (Trifolium spp.), plantains (Plantago spp.), ryegrass (Lolium perenne L.), the tobacco plant (Nicotiana tabacum L.), leek (Allium porrum L.), Sudan grass (Sorghum bicolor (L.) Moench), corn (Zea mays L.) and bahia grass (Paspalum notatum Flugge).

Pasteurization, steaming and/or irradiation are necessary to avoid contamination of the growing media. The use of a well-aerated substrate is also recommended. The manufacturer must provide the customer who intends to introduce the AMF inoculum to a target plant with basic information and assistance concerning its chemical and physical characteristics, such as nutrient content, pH and salinity. In particular, when elevated quantities of inoculum are used in agricultural fields, or in a pot-culture, controlling the nutrient content is of crucial impor‐ tance, as it might lead growers to rethink their normally adopted fertilization practices. Conventionally, inoculum formulation processing consists of sieving the substrate and chopped roots of the trap plant in order to retrieve AMF propagules that can be included in the inoculum. This means that the carry-over of a certain amount of nutrients to the final product is unavoidable. Nevertheless, if trap plant pots are not over-fertilized, as it should be during inoculum formulation, the nutrient content will be negligible. A solution to the problem could be the laborious approach of completely separating the spores, mycelium and colonized trap plant root fragments from the used growing media. These substrate-free propagules could then be mixed with an inert-like carrier at a desired rate. The amendment of the inoculum should be compatible with the AMF, almost inert and only serve to support mycorrhizal development. Optimum P and N, but also other macroelement levels, have to be tuned to 176 Biodiversity - The Dynamic Balance of the Planet

specific plant–AMF combinations, as mentioned in the previous section, in order not to reduce AMF propagation and diminish plant dependency on mycorrhization after inoculation. Other edaphic factors, such as pH, salinity, soil temperature, moisture and soil aeration, should also be controlled to optimize AMF inoculation. Since the inadequacy of the nutrient composition dramatically affects AMF development, conventional soil analyses should be performed on the formulated inoculum, in independent official laboratories, as a quality control step. This way, the manufacturer will be provided with a certificate that guarantees the customers the validity of the data reported on the label and, therefore, enhances the quality of the inoculum. During experimental tests on the beneficial effects of inoculants, researchers often adopt an important practice in order to be able to differentiate between the effects of the inoculum carrier and the AMF portion, i.e. the use of a sterilized inoculum as a control, the so-called “mock” inoculum [145]. This practice of including a non-inoculated and a “mock” inoculated control should be considered by end-users who are willing to assess the eventual beneficial effect of AMF inoculation.

A few alternatives to the pot-culture method are available, regarding inoculum production and formulation. Other soilless culture systems, such as aeroponics and hydroponics, enable the production of pure clean spores and maximize growing conditions for the host plant [146]. Aeroponic inoculum production has long been scientifically validated [147,148], and could soon reach massive commercialization levels. Root-organ monoxenic culture is another method that allows the successful large-scale propagation of AMF which can be used directly as an inoculum. Unfortunately, the protocol for this method of propagation is not easily adjustable to all AMF strains. So far, several dozens of AMF species and strains have been propagated in vitro with the right synthetic growth medium and growth conditions. This type of culture consists of AMF inoculated excised roots (often Daucus carota L.) that have acquired the ability to uncontrollably proliferate, without the epigeous portion, after transformation with an Agrobacterium rhizogenes Conn. strain. This method of propagation does not require high specialization, and facilitates the control of AMF strain purity. As mentioned before, it is suitable for large-scale production, as a massive number of spores (several thousand), mycelium and colonized roots [149] can be obtained from one Petri dish in just 4 months, and from the consecutive subcultures [150]. AMF propagated with this technique have been shown to successfully re-colonize plant roots [151,152]. A possible further advantage of the AMF inoculum production process could be the use of bioreactors with liquid transformed root- organ cultures aimed at the large-scale propagation of AMF [153]. These tools may become suitable for commercialization in the near future and will lead to reduced labor and enhanced automation. However, as the AMF are produced in association with transformed roots, the product will only be intended for research use and may not be used for open-field inoculation.

The final product could become available on the market as a powder or granular substrate made from mixed inert-like materials, such as peat, compost, vermiculite, perlite, quartz sand, micronized zeolite and expanded clay, where colonized root fragments (1-5 mm long), spores and hyphal networks are uniformly distributed. Liquid inocula, dedicated to horticultural use, obtained from a hydroponic culture, or from a spore/mycelium suspension in a liquid carrier, represent a possible alternative final product [154]. As a final step before commercialization, Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management 177 http://dx.doi.org/10.5772/58231 the AMF composition should be characterized in order to control inoculum purity and to trace the inoculated strains. This prevents poor quality inocula from being put on the market.

The storage methodologies should preserve a product’s high and consistent quality, and be simple and inexpensive at the same time. AMF viability and efficiency can be maintained for several months at room temperature (20-25°C), but the inocula must be kept in their packaging and must be partially dried. The main inconvenience that could occur during the storage period is that spores can sometimes become dormant, thus decreasing germination rates drastically [155]. However, a cold-storage period could be used to break dormancy [156]. Longer-term storage of liquid or dry inocula could be conducted at 5°C for both in vivo and in vitro propagated AMF [127]. Research culture collections are often stored using more sophisticated and expensive preservation techniques. These include the maintenance of monospecific inocula on living host plants (with regular molecular checks regarding the AMF identity), or alginate bead mediated encapsulation-drying and cryopreservation [157,158].

4. Perspectives

Future research in this field will have to concern the formulation of AMF isolate collections, with comprehensive information on host-preference, edaphic and climatic adaptation, and stress and disturbance tolerance. This will help manufacturers address their product towards different uses, including agricultural use, as well as new fields of application, such as the green architecture of urban sites [159]. At the same time, farmers will have to begin asking for assistance from experts in the field when introducing AMF to their cropping systems. Scientists should also carry out large-scale multi-location field trials, and conduct cost-benefit analyses, in order to increase awareness among the end-users of AMF inocula.

By 2050, global agriculture will have the task of doubling food production in order to feed the world [160]. At the same time, dependence on inorganic fertilizers and pesticides must be reduced. For these reasons, significant advances in AMF research are needed to allow their stable use in agriculture. Their application and synergistic combination with other functionally efficient microbial consortia that include PGPR (Plant Growth Promoting Rhizobacteria), saprophytic fungi and other helper microorganisms [161], will help farmers develop a more sustainable cropping system.

Acknowledgements

Our work was financially supported by the following institutions: Piemonte Region (ECO‐ FLOR and PRO-LACTE projects), Alcotra (FIORIBIO2 project), and EU (PURE project). The authors would like to thank Dr. Valentina Scariot for her coordinating work in the ECOFLOR project and Lucia Allione for her support in the funding management of the projects. 178 Biodiversity - The Dynamic Balance of the Planet

Author details

Andrea Berruti1, Roberto Borriello1, Alberto Orgiazzi2, Antonio C. Barbera3, Erica Lumini1 and Valeria Bianciotto1

1 National Research Council, Plant Protection Institute – Turin UOS, Torino, Italy

2 European Commission, Joint Research Centre, Institute for Environment and Sustainability, Ispra (VA), Italy

3 DISPA, University of Catania, Catania, Italy

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OPEN Dissection of niche competition between introduced and indigenous arbuscular mycorrhizal Received: 8 November 2017 Accepted: 26 April 2018 fungi with respect to soybean yield Published: xx xx xxxx responses Rieko Niwa1,7, Takuya Koyama2,8, Takumi Sato3, Katsuki Adachi2, Keitaro Tawaraya 3, Shusei Sato4,5, Hideki Hirakawa5, Shigenobu Yoshida1 & Tatsuhiro Ezawa 6

Arbuscular mycorrhizal (AM) fungi associate with most land plants and deliver phosphorus to the host. Identifcation of biotic/abiotic factors that determine crop responses to AM fungal inoculation is an essential step for successful application of the fungi in sustainable agriculture. We conducted three feld trials on soybean with a commercial inoculum and developed a new molecular tool to dissect interactions between the inoculum and indigenous fungi on the MiSeq sequencing platform. Regression analysis indicated that sequence read abundance of the inoculum fungus was the most signifcant factor that determined soybean yield responses to the inoculation, suggesting that dominance of the inoculum fungus is a necessary condition for positive yield responses. Agricultural practices (fallow/cropping in the previous year) greatly afected the colonization levels (i.e. read abundances) of the inoculum fungus via altering the propagule density of indigenous AM fungi. Analysis of niche competition revealed that the inoculum fungus competed mainly with the indigenous fungi that are commonly distributed in the trial sites, probably because their life-history strategy is the same as that of the inoculum fungus. In conclusion, we provide a new framework for evaluating the signifcance of environmental factors towards successful application of AM fungi in agriculture.

Arbuscular mycorrhizal (AM) fungi that belong to the subphylum Glomeromycotina form mutualistic associations with about 80% of land plants, including important agricultural crops, and deliver mineral nutrients, especially phosphate, to the host1. Potential of AM fungi in the improvement of crop yield, therefore, has been studied in the context of sustainable agriculture2–5. Soybean (Glycine max (L.) Merrill.) is the most widely grown leguminous crop that provides an important source of protein and oil. Recently, increases in yield by inoculation of AM fungi in feld trials have been reported in soybean6–8. A meta-analysis of the published studies on 8 legume crops revealed that, although ‘strict’ non-mycorrhizal control was absent under the feld conditions, the changes in yield by AM fungal inoculation varied from −4% to +24% and was only +9% on average, which was marginal compared with those in the pot experiments (45% increase in average)9. Accordingly, identifcation of biotic and abiotic factors that determine soybean responses to AM fungal inoculation in the feld is an essential step for successful application of AM fungi in agricultural production.

1Central Region Agricultural Research Center, National Agriculture and Food Research Organization (NARO), 2-1-18 Kannondai, Tsukuba, 305-8666, Japan. 2Kyushu Okinawa Agricultural Research Center, NARO, 6651-2 Miyakonojo, Miyazaki, 885-0091, Japan. 3Faculty of Agriculture, Yamagata University, Tsuruoka, 997-8555, Japan. 4Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577, Japan. 5Kazusa DNA Research Institute, Kisarazu, 292- 0818, Japan. 6Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. 7Present address: Institute for Horticultural Plant Breeding, 2-5-1 Kamishiki, Matsudo, Chiba, 270-2221, Japan. 8Present address: School of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi, 321-8505, Japan. Rieko Niwa and Takuya Koyama contributed equally to this work. Correspondence and requests for materials should be addressed to T.E. (email: [email protected])

Scientific REPOrtS | (2018)8:7419 | DOI:10.1038/s41598-018-25701-4 1 www.nature.com/scientificreports/

One crucial biotic factor is indigenous AM fungi; they are well adapted to the local environment10 and thus competitive with introduced (inoculum) fungi4. Various agricultural practices/management alter indigenous AM fungal community qualitatively and quantitatively. Te long-term application of chemical fertilizer11,12 and till- age13 have a signifcant impact on the community compositions. Organic farming increases species richness of the fungi, compared with that in conventional agriculture14,15. Cropping of AM fungal host plants improves growth of succeeding crops via increasing indigenous AM fungal population16. It has been suggested that these factors afect the efectiveness of AM fungal inoculation17. For example, long-term fallow decreases the propagule density of indigenous AM fungi and increases the responsiveness of leek to AM fungal inoculation18, suggesting that the inoculation of the fungi is more efective under conditions where population of indigenous fungi is smaller or they perform poorer. Little is known, however, about how agricultural practices/management afect the responsiveness of plants to the inoculation via altering the interactions between indigenous and introduced AM fungi. Specifc molecular markers for tracking introduced AM fungi in the feld have been developed. Two strains of non-native Funneliformis mosseae were successfully traced and discriminated from native strains of the species by PCR-RFLP targeting the large subunit (LSU) ribosomal RNA gene (rDNA)2. In Rhizophagus irregularis genotype-specifc markers targeting simple sequence repeats, a nuclear gene intron, and mitochondrial LSU rDNA introns have also been developed19. To dissect the interactions between introduced and indigenous fungi, however, not only particular fungal strains but also indigenous AM fungi should simultaneously be detected on the same platform. Te rDNA transcription unit, including the small subunit (SSU), internal transcribed spacer (ITS), and LSU, has most commonly been employed for dissecting AM fungal community. In this approach, taxonomic resolution of the fungi greatly depends on the combination of the target region of rDNA and sequencing platform. On the Sanger (clone library/random sequencing) and Roche 454 platforms, sequencing of the SSU rDNA, followed by the assignment of the sequences to the virtual taxa in the MaarjAM database20,21, is the most common approach22. Recently, Schlaeppi et al.23 demonstrated that introduced AM fungi were traceable in the feld via sequencing a 1.5-kbp region spanning the SSU rDNA, ITS, and LSU rDNA on the medium-throughput sequencing platform PacBio. Whereas Illumina MiSeq generates short (300 bp) but a massive number (up to 50 M) of paired-end reads, which is a great advantage for community ecology, and in fact, the platform has become standard in bacterial ecology24,25. To take this advantage in AM fungal ecology, we chose the LSU rDNA as a target for MiSeq sequencing. In AM fungi, the sequence variations in the LSU rDNA provide high-resolution taxonomic information26 and have widely been employed in the ecology11,27–30. Within the LSU rDNA, in particular, the divergent domain 2 (D2) that is less than 400 bp in length is short enough to cover by MiSeq 300-bp paired-end sequencing and provides sufcient taxonomic resolution31. Here, we provide a framework for identifying biotic and abiotic factors that determine the responsiveness of soybean to AM fungal inoculation in the feld through tracking an inoculum AM fungus in indigenous community, in which the following two hypotheses were addressed; (i) the dominance of inoculum fungus in the host roots is a necessary condition for positive yield responses, and (ii) the propagule density of indigenous AM fungi largely determines the extent of the colonization of inoculum fungus. To test these hypotheses, we developed molecular tools adapted to the MiSeq sequencing platform, including a database for taxonomic assignment and a data processing pipeline driven via an open web interface, for high-throughput community analysis of the fungi. Materials and Methods Fungal inoculum. An AM fungal inoculum Glomus sp. strain R-10 (R-10) was purchased from Idemitsu Kosan Co., Ltd., Tokyo. Te inoculum consists of spores and root fragments of R-10 with a crystalline-silica carrier. For the control treatments, we obtained the carrier that was free of the propagule (i.e. unprocessed carrier) from the manufacturer. Te most probable number (MPN) of the inoculum was 14 propagules g−1, which was determined prior to the feld trials as described in Supplementary Methods S1. To defne a DNA tracking marker for R-10, the inoculum was mixed with sterilized soil at a rate of 50 g kg−1 soil, and Allium fstulosum cv. Motokura was grown in nursery pots in a greenhouse. Afer 42 days, the roots were harvested, freeze-dried, and stored at −30 °C for DNA extraction and sequencing.

Field trials and sampling. Tree trials were designed in adjacent feld sites in Kyushu Okinawa Agricultural Research Center, National Agricultural and Food Research Organization, Miyakonojo, Miyazaki, Japan (31°45′05′′N 131°00′46′′E). In the preceding year 2014, trials 1 and 2 were bare fallowed (T1_BF and T2_BF), whereas palisade grass Brachiaria brizantha (Hochst. ex A. Rich.) cv. MG5 was grown from July to Sep in trial 3 (T3_PG). Te felds for T1_BF and T2_BF were periodically plowed to control weeds in 2014. Details of the environmental conditions are described in Supplementary Methods S2. In 2015 soil samples were collected from each replicated block (n = 4) prior to fertilizer application to determine the MPN of AM fungal propagule (Supplementary Methods S1). All these trial sites have a history of soybean cultivation, and thus nitrogen-fxing nodules are formed by native rhizobia (i.e. without rhizobial inoculation). Phosphorus (P) fertilizer was applied −1 at three diferent levels, 0 (P0), 50 (P50), and 100 (P100) kg P2O5 ha , in T1_BF and at two diferent levels, −1 0 (P0) and 100 (P100) kg P2O5 ha , in T2_BF and T3_PG. Nitrogen (N) and potassium (K) fertilizers were −1 −1 applied at 40 kg N ha and 120 kg K2O ha , respectively, in all trials. Five grams of either the inoculum or the propagule-free carrier was placed at a depth of 80 mm and buried, and then three seeds of Glycine max (L.) Merrill. cv. Fukuyutaka were sown on 27 and 28 July in T1_BF and on 24 July in T2_BF and T3_PG and thinned to two plants afer the frst trifoliate appeared. Te replicate plot size in Trial 1 (T1_BF) was 3.9 × 4.4 m, and that in Trial 2 and 3 (T2_BF and T3_PG) was 3.9 × 2.8 m, in which six rows were arranged with distance of 0.65 m between rows (0.2 m between hills). Weed in the plots were controlled manually. Intertillage and ridging were conducted in the middle of Aug. Te treatments were arranged in the randomized complete block design. At the time of fowering (Aug 31–Sep 4), roots and root-zone soils (20 × 20-cm square, 25 cm in depth) were collected from four plants in each plot and combined. Afer gentle washing of the roots with tap water, about 1 g of

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Figure 1. Relative locations and directions of PCR primers for amplifcation of the large subunit ribosomal RNA gene. Tree forward primers, FLd1, FLd2 and FLd3, were designed in the region between the divergent domains 1 (D1) and 2 (D2).

subsamples (lateral roots attached to the tap root) were collected in the middle of the tap root, cut into 1-cm segments, randomized in water, blotted on a paper towel, freeze-dried, and stored at −30 °C for DNA extraction. Te soil samples were dried in greenhouse, passed through a 2-mm stainless sieve, and stored at room temperature for chemical analysis. Te above ground part was harvested on 11 Nov in T1_BF and on 24 Nov in T2_BF and T3_PG from 24 plants grown within 1.56 m2 (1.3 × 1.2 m) in each plot, and grain yield of 15% moisture content was recorded afer drying at 80 °C for 72 h. For measuring total biomass and P concentration of the above ground part, another four plants were harvested in each plot, combined, dried at 80 °C for 72 h, weighed, ground with a mill, and stored in a desiccator.

Soil and plant analyses. Soil pH (H2O) was measured at a 1: 2.5 soil: water ratio (w/v) using an electrode afer shaking for 1 h at 160 rpm. Total carbon (C) and N were analyzed with a CNS analyzer. Available phosphorus was extracted according to Truog (1930)32 with a modifcation in the extraction bufer11 and measured colori- metrically. Exchangeable calcium (Ca), magnesium (Mg), and K were displaced with 1 M ammonium acetate and measured with atomic absorption spectroscopy (Ca and Mg) and fame photometry (K). Cation exchange capacity was determined by summation of exchangeable base cation and exchangeable acidity. Nitrate and ammonia concentrations were determined by the hydrazine reduction and indophenol methods, respectively. Phosphate absorption coefcients were measured by the ammonium phosphate methods. Te ground plant samples were digested with a mixture of nitric acid-perchloric acid according to Miller (1998)33, and P concentration in the digests was determined by the vanadomolybdate-yellow assay34.

DNA extraction and PCR amplifcation. Te freeze-dried root samples were ground in 3-mL tubes with a metal cone at 3,000 rpm for 45 s at room temperature using Multi-Beads Shocker (Yasui Kikai, Osaka), and DNA was extracted from the ground samples (10–20 mg) with DNeasy Plant Mini Kit (Qiagen, Tokyo) according to the manufacturer’s instructions, and stored at −30 °C. Tree mixed PCR primers, FLd1, FLd2, and FLd3, were designed in the conserved region between the D1 and D2 in the LSU rDNA (Fig. 1 and Supplementary Table S1), and their performance was assessed prior to the main experiment by comparing with the eukaryote-universal primer LR135 in combination with the fungi-specifc reverse primer FLR236 (Supplementary Methods S3). In the main experiment the region was amplifed in a 25-µL reaction mixture of Expand High-Fidelity PLUS PCR System (Roche Diagnostics, Tokyo), 0.5 µM of the primers, and fve diferent amounts of DNA template (0.01, 0.02, 0.1, 0.5, and 1.0 µL per 25 µL for each sample) using C1000 Touch Termal Cycler (BIO-RAD, Tokyo) with the following program: initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 15 s, annealing at 48 °C, polymerization at 72 °C for 1 min, and fnal extension at 72 °C for 10 min. A preliminary experiment showed that the relative abundances of diferent AM fungal sequences in the PCR products raised from diferent amounts of DNA template difered from each other. Accordingly, all PCR products obtained from the fve diferent amounts of DNA template were combined, purifed with Agencourt AMPure XP PCR Purifcation System (Beckman Coulter, Tokyo), and subjected to sequencing.

Sequencing and data processing. Te frst PCR products were subjected to the 2nd PCR to attach dual indices and Illumina sequencing adaptors using Nextera XT Index Kit v2 (Illumina, Tokyo) according to the 16 S Metagenomic Sequencing Library Preparation protocol (https://support.illumina.com/downloads/16s_metagen- omic_sequencing_library_preparation.html). Te libraries were pooled, adjusted to 4 nM DNA, denatured with NaOH, and then diluted with the hybridization bufer to the fnal concentration of 6 pM DNA, and 300-bp paired-end sequencing was carried out by the Illumina MiSeq platform using MiSeq Reagent Kit v3 (600 Cycles) (Illumina K.K., Tokyo). Te nucleotides with a quality value (QV) <30 in the 3′ terminal and the adapter-index sequence in the 5′ terminal were trimmed from the MiSeq reads by PRINSEQ v0.20.437, and those shorter than 200 bp were excluded. Afer the quality fltering, an overlap fragment of the 300-bp-paired reads (read 1 and read 2) were constructed by using COPE v1.1.338 with the minimum and maximum overlap lengths of 10 and 300 nt, respectively. Te merged sequence reads were subjected to BLASTN searches against reference sequences (database) composed of 412 operational taxonomic units (OTUs) of glomeromycotinan (AM) fungi defned in this study (Supplementary Methods S4, Table S5, and Fig. S1) and 82,208 sequences of non-glomeromycotinan fungi obtained from Ribosomal Database Project39, in which sequence reads similar to an AM fungal OTU and those similar to a non-glomeromycotinan species were assigned to the OTU/species with diferent criteria as follows. For the reads similar to AM fungal OTUs, only those that met the criteria of E-value ≤ −100, ≥95% nt identity,

Scientific REPOrtS | (2018)8:7419 | DOI:10.1038/s41598-018-25701-4 3 www.nature.com/scientificreports/

and alignment length ≥330 bp with one of the AM fungal OTUs were assigned to the OTU. Whereas, for the reads similar to non-glomeromycotinan sequences, only those that met the criteria of E-value ≤ −100, ≥95% nt identity, and alignment length ≥220 bp with one of the 82,208 sequences were assigned to the species. All these analyses were executed in the open web interface “Arbuscular mycorrhizal fungi classifcation pipeline” (http:// amfungi.kazusa.or.jp) constructed in this study.

Statistical analysis. All statistical analyses were performed with R 3.2.340. Two-way analysis of variance (ANOVA) with random effects for block differences were applied to evaluate the effects of the inoculation and P fertilizer application on shoot P concentration and grain yield. Vegan package for R41 was employed for β-diversity analysis with Bray-Curtis similarity index (read-abundance data based index) and for permutation multivariate analysis of variance (PERMANOVA) in which Bray-Curtis index was used as a measure of similarity (9999 permutations). In regression analyses all OTUs obtained by sequencing of the inoculum fungus R-10 were combined as ‘R-10-type OTUs’, in which ‘read abundance of the R-10-type OTUs’ represents the sum of the sequence reads assigned to the OTUs. Simple linear regression models were applied to analyze correlations between read abundances of the R-10-type OTUs (log-transformed) and MPNs of indigenous AM fungal propagule (log-transformed) and between the read abundances and soybean yield responses to the inoculation. Te yield responses were calculated by the equation (1) as follows:

Yieldresponsetoinoculation =−(Yield in inoculated plot Mean yieldincontrol plots)/Meanyield in controlplots (1) Te multiple linear regression model was applied to evaluate to the efects of the read abundance of R-10-type OTUs and the environmental factors (Supplementary Table S6) on the yield responses to the inoculation. Te best model was selected with reference to Akaike’s information criterion (AIC)42 calculated by the stepwise method using MASS package in R. Te logistic regression model with random efects for block diferences was applied to evaluate the efect of environmental factors on the read abundance of R-10-type OTUs using glmmML package in R43. Te best model was selected with reference to AIC calculated using MuMIn package. To dissect competition between the introduced (inoculum) and indigenous AM fungi, the following two indices were employed. ‘Commonness’ that is the index referred to as niche breadth in Levins (2013)44 and Pandit et al.45 was employed to evaluate their habitat specialization and calculated for each indigenous OTU by the equa- tions (2 and 3) as follows:

= n 2 Commonness 1/∑(0i= )Pij (2)

Mean read number of OTUijintrial Pij = Sumofmeanreadnumbers of OTUij neachtiral (3) where only the read number data in the uninoculated control were used to compute. OTUs with a higher value of commonness distribute more evenly across the habitats and thus are considered to be habitat generalists45. Rare OTUs of which the mean relative abundance was less than 0.2% of total read were not considered in this analysis. Robustness of each indigenous OTU against the introduction of R-10 fungus was defned as a ratio of the mean read abundance in the inoculated plots to that in the control plots in each trial.

Data availability. All data used in this study are disclosed in the paper and supplementary information fles. Te raw data are available from the corresponding author on reasonable request. Results Validation of new primer for MiSeq platform. Approximately 350–450 bp fragments of the D2 were successfully amplifed from the DNA extract of the maize test sample both with FLd1 and FLd3 primers, but not with FLd2 primer (data not shown). AM fungal OTU compositions of these PCR products (sequenced by the Sanger method) were similar to each other and also similar to that amplifed with the eukaryote-universal primer LR1 (Supplementary Table S7). Whereas most of the sequences obtained with FLd3 were assigned to AM fungal OTUs (>88%), comparable to those obtained with LR1 (>83%), but only a half of the sequences obtained with FLd1 could be assigned to AM fungal OTUs. Accordingly, we chose FLd3, the mixture of FLd3-1, -2, -3, and -4 (Supplementary Table S1), and further assessed its performance on the MiSeq platform. Te D2 region was amplifed from four test samples with FLd3 and sequenced, and the reads were separated into four groups according to their 5′-primer sequence prior to OTU assignment to test whether the diferent primers amplify different AM fungal sequences as expected. Te OTU compositions obtained with the four primers, however, were highly similar to each other (Supplementary Table S8); Bray-Curtis similarity indices between the communities obtained from the same DNA template with the diferent primers were consistently higher than 0.9 across all combinations (Supplementary Table S9). In addition, the numbers of the sequence read assigned to an AM fungal OTU were not markedly diferent among the four primers. Tese results suggest that mixing of the four primers diferentiates the OTU compositions only marginally and that efciencies of these primers in the amplifcation of D2 are not diferent. We therefore decided to employ FLd3-1 as a forward primer, because its sequence covers the majority of glomeromycotinan LSU rDNA.

Soybean responses to inoculation with respect to biotic and abiotic factors. MPNs of AM fungal propagule in T1_BF and T2_BF were around 0.02 propagule mL soil−1, but that in T3_PG was about an order of magnitude higher (Fig. 2). Levels of available phosphate were signifcantly higher in T2_BF and T3_PG than in

Scientific REPOrtS | (2018)8:7419 | DOI:10.1038/s41598-018-25701-4 4 www.nature.com/scientificreports/

Figure 2. Most probable numbers (MPNs) of AM fungal propagule in the three trial sites. Soil samples were collected from four replicated plots in each trial site prior to fertilizer application and mixed with autoclaved sand to obtain a dilution series of 2−1, 2−3, 2−5, and 2−7, and Lotus japonicus was grown in the soil mixtures in a growth chamber for 26 days. MPNs were estimated based on the presence/absence of fungal colonization recorded under a dissecting microscope. Te vertical bars indicate standard errors (n = 4). Te diferent letters indicate signifcant diferences among the three trial sites (Tukey HSD test, p < 0.05).

Trial 1_Bare fallow Trial 2_Bare fallow Trial 3_Palisade grass P application Shoot P Yield Shoot P Yield Shoot P Yield (kg ha−1) Inoculum (mg P g−1 DW) (kg ha−1) (mg P g−1 DW) (kg ha−1) (mg P g−1 DW) (kg ha−1) Control 4.06 ± 0.09a 3079.9 ± 76.7 4.26 ± 0.10 3393.8 ± 41.6 4.40 ± 0.07 3192.6 ± 52.7 0 R-10 4.17 ± 0.09 3273.1 ± 15.3 4.34 ± 0.09 3171.1 ± 136.4 4.36 ± 0.02 2811.6 ± 118.4 Control 4.10 ± 0.05 3111.1 ± 46.5 — — — — 50 R-10 4.20 ± 0.05 3220.4 ± 65.6 — — — — Control 4.06 ± 0.04 3081.4 ± 105 4.31 ± 0.09 3061.3 ± 148.7 4.60 ± 0.26 3166.3 ± 95.1 100 R-10 4.30 ± 0.05 3125.5 ± 46.1 4.29 ± 0.05 3266.5 ± 83.6 4.38 ± 0.06 3103.9 ± 56.4 P nsb ns ns ns ns ns Inoculation ** * ns ns ns * P × Inoculation ns ns ns ns ns ns

Table 1. Efect of AM fungal inoculation and phosphorus (P) fertilizer application on shoot P concentration of the fowering stage and grain yield of Glycine max in the three trials. a±SE (n = 4). bANOVA: ns, not signifcant; *P < 0.05; **P < 0.01.

T1_BF, but P-fertilizer application did not diferentiate the levels in all trials (Supplementary Table S6). Levels of exchangeable Ca and Mg were also slightly higher in T2_BF and T3_PG. Te inoculation of R-10 signifcantly increased shoot P concentration in the fowering stage and grain yield in T1_BF, but signifcantly decreased grain yield in T3_PG (Table 1). In T2_BF the inoculation did not afect shoot P concentration and grain yield. No signifcant efects of P-fertilizer application and its interaction with the inoculation on yield and shoot P concentration were observed in all trials. Root nodule formation was observed in all plants. Te inoculum sequencing revealed that R-10 fungus consists of 21 OTUs (Supplementary Table S10), all of which are likely to belong to Rhizophagus irregularis, except for the OTU 382_Rhz that is related to R. intraradices (Supplementary Fig. S2). Tese OTUs were employed for a tracking marker in the trials. In sample sequencing, 15,000 to 30,000 high-quality reads of AM fungi were obtained for each sample and normalized to 10,000 reads per sample for community analysis. In the uninoculated control plots the R-10-type OTUs were present up to 30% of total AM fungal read (Fig. 3 and Supplementary Table S10). In T1_BF and T2_BF, however, the inoculation of R-10 drastically increased the read abundances of R-10-type OTUs to more than 70% of total read, suggesting that R-10 fungus largely dominated the communities in these trials. In contrast, the inoculation had a limited impact on the communities in T3_PG. In multiple linear regression analysis on the relationship of environmental (biotic and abiotic) factors with the yield responses to the R-10 inoculation, the best ft model with the lowest AIC indicated that the read abundances of R-10-type OTUs is the most signifcant factor (R = 0.002, p < 0.001) of which a standardized partial regression coefcient was 0.67 (Table 2). In fact, the read abundances of R-10-type OTUs were positively correlated with the

Scientific REPOrtS | (2018)8:7419 | DOI:10.1038/s41598-018-25701-4 5 www.nature.com/scientificreports/

Figure 3. Average relative read abundance of the R-10-type operational taxonomic units (OTUs) in total AM fungal sequence read in the soybean root samples in the uninoculated (control) and inoculated (R-10) plots of the three trials (n = 4). Te black and gray bars represent the relative read abundance of R-10-type and other OTUs, respectively.

Standardized partial Variable Coefcient estimate Standard error P value regression coefcient Read abundance of R-10-type OTUs 0.00205 0.00046 0.00019 0.67 P application level 0.00033 0.00023 0.16 0.21 Phosphate absorption coefcient 0.00013 0.00008 0.12 0.24

Table 2. Efect size of biotic and abiotic factors on yield response to R-10 inoculation estimated by multiple linear regression analysis.

Coefcient Variable estimate Standard error P valuea Odds ratio P application level 0.0041 0.0013 0.0013 1.00 Phosphate absorption coefcient −0.0019 0.00091 0.037 1.00 MPNb −8.68 2.01 1.5.E-05 0.00017 Nitrate nitrogen 0.63 0.30 0.035 1.87 Exchangeable potassium −0.097 0.04 0.023 0.91

Table 3. Efect size of biotic and abiotic factors on the read abundance of R-10-type OTUs estimated by logistic regression with a random efect model. aNeyman-Pearson test. bMost probable numbers of AM fungal propagule.

yield responses to the inoculation (R2 = 0.37, p < 0.001) (Supplementary Fig. S3), supporting the frst hypothesis. In logistic regression analysis on the factors afecting the R-10 read abundances, the best ft model with the lowest AIC indicated that MPN is the most signifcant factor (R = −8.68, p < 0.001) of which an odds ratio was 0.00017, implying that the read abundances would be decreased by 0.00017-fold with increasing MPN by 1 (Table 3). In fact, the read abundances of R-10-type OTUs in the inoculated plots were negatively correlated with the MPNs (R = −0.73, p < 0.001) (Supplementary Fig. S4), supporting the second hypothesis.

Interactions between introduced and indigenous AM fungi. Interactions between the inoculum and indigenous AM fungi were frst dissected via assessing the impact of inoculation on the community compositions. Two-way PERMANOVA indicated that the community compositions were diferent among the three trial felds and that the inoculation, as well as the interaction, had a signifcant impact on the compositions (Supplementary Table S11). Bray-Curtis similarity index between the inoculated and control plots was signifcantly higher in T3_ PG than in T1_BF and T2_BF (Fig. 4). Tese results imply that the impact of R-10 inoculation on the community

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Figure 4. Box plots of Bray-Curtis similarity index between the inoculated and uninoculated communities in the three trials. Te bottom, top, and median lines of the boxes represent the frst quantiles, third quantiles, and medians, respectively, and the error bars represent the range of the data. Te diferent letters indicate signifcant diferences among the three trials (Steel-Dwass test, p < 0.05).

compositions was smaller in T3_PG; that is, the indigenous communities in T3_PG were more robust against the inoculation than those in T1_BF and T2_BF. To characterize the indigenous fungi that compete with R-10 fungus, the two indices commonness and robustness against the inoculation were calculated for each indigenous fungal OTU (Supplementary Table S12), except for the R-10-type OTUs, because the OTUs originate from R-10 fungus could not be discriminated from those present in the indigenous communities. Te values of commonness were rather negatively correlated with those of robustness in T1_BF (R = −0.50, p = 0.018) and T2_BF (R = −0.29, p = 0.12), implying that more common fungi were generally less robust against the introduction of R-10 fungus in these trial sites (Fig. 5). In contrast, there was a positive correlation between commonness and robustness in T3_PG (R = 0.72, p < 0.001); more common fungi were more robust against the introduction of R-10 fungus. Tese results imply that R-10 fungus competed for the niche mainly with the fungi that are commonly distributed in the trial sites. Discussion It has been proposed that abundance of indigenous AM fungi18,46, soil fertility4, and crop rotation16, as well as plant and fungal genotypes47,48, are the major factors that determine inoculation success. In these studies, however, the colonization of inoculum fungi could not be taken into account due to technical difculties. In the present study, not only the environmental factors but also the interactions between the inoculum and indigenous fungi were incorporated in the modeling of the soybean yield responses to AM fungal inoculation, which provides a new framework for evaluating the signifcance of environmental factors towards successful application of the fungi in agricultural production. Recently, technical feasibility for tracking AM fungal inocula in feld trials has been demonstrated2,19,23,49, but methods that enable to analyze interactions between introduced and indigenous fungi on high-throughput sequencing platforms have not yet been proposed. In this study, no inoculum-specifc OTUs, that is, those that were absent in the feld, but abundant in the inoculum fungus, were found, which implies that increases in the inoculum OTUs should be interpreted carefully. In the inoculated plots of T1_BF and T2_BF, the drastic increases in the read abundance of R-10-type OTUs was observed, implying that the inoculum fungus was highly likely to dominate in these plots. It was not possible, however, to estimate what proportion of the sequence reads was of the inoculum fungus. Accordingly, it is considered that the present method could be applicable only if (i) inoculum fungi have specifc OTUs or (ii) dramatic increases in inoculum fungal OTUs by inoculation are observed in the presence of the same OTUs in indigenous community. Our modeling indicated that the read abundances of inoculum OTUs in the fowering stage is the most important factor that determine the soybean yield responses to the inoculation. Tese observations strengthen the fndings in the meta-analysis50 that demonstrated that rapid colonization and dominance of inoculum fungi in early growth stages are essential to obtain positive yield responses to AM fungal inoculation. In the case of soybean, it seems also likely that the positive yield responses were not only due to the direct efect of the inoculation but also due to an indirect efect of the inoculation, such as enhancement of rhizobial N fxation through improving P nutrition. It has been well documented that N fxation in the nodules is largely limited by P nutrition even under conditions of adequate P fertilization (reviewed in Graham and Vance51). Tis idea further raises the following two questions; (i) whether the efectiveness of AM fungal inoculation could be enhanced under conditions of

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Figure 5. Correlation analysis between commonnesses and robustnesses of indigenous fungal OTUs in T1_BF (circles), T2_BF (triangles), and T3_PG (squares). Commonness that was referred to as niche breadth was calculated according to Pandit et al.45 to evaluate their habitat specialization, and robustness against the inoculation of R-10 fungus was calculated as a ratio of the mean read abundance in the inoculated plots to that in the control plots. Pearson correlation coefcients (R) were −0.50 (p = 0.018), −0.29 (p = 0.12), and 0.72 (p < 0.001) in T1_BF, T2_BF, and T3_PG, respectively.

abundant rhizobia and (ii) whether AM fungal inoculation could reduce N fertilizer input via enhancing N fxation without reduction in yield. Although levels of nodule formation (e.g., nodule number and weight) were not evaluated in the present study, it would be worthwhile to incorporate these factors for the modeling. Unexpectedly, increases in shoot P concentration and grain yield were not observed in the inoculated plots of T2_BF in which the large dominance of R-10-type OTUs was observed. Tese observations suggest that read abundances of inoculum fungal OTUs would not be a universal indicator to predict the efectiveness of inoculation and, further, that more comprehensive analysis on environmental factors, together with the read abundances of inoculum OTUs, is necessary. In T2_BF it seems likely that the high levels of available phosphate in the soil cancelled the beneft of inoculation (e.g., Tawaraya et al.52; Verbruggen et al.4), although the multiple linear regression analysis indicated that available phosphate was not a signifcant factor for the responsiveness of soybean. More feld trials in a wider range of environments are required to improve reliability of the modeling.

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Te levels of the colonization (i.e. read abundances) of inoculum fungus were negatively correlated with the propagule density of indigenous AM fungi, although these data were obtained from only three trials. It has been suggested that indigenous AM fungal population can be maintained by cropping of AM plants, but is decreased by non-host cropping53 and bare fallow18,46. In addition, the growth/yield gap afer bare fallow could be recovered by the reintroduction46 or inoculation18 of AM fungi. Our results support these fndings via analyzing the interactions between the introduced and indigenous fungi. Further trials, however, should be conducted in arable felds with various agricultural practices/management in diverse environments to confrm the signifcance of propagule density in the colonization of introduced fungi. In agricultural ecosystems frequent disturbance (e.g., tillage) may act as selection pressure for the fungi that have the life history strategy of ‘ruderal’54, that is, those that colonize rapidly55, regenerate hyphal networks ef- ciently afer disturbance56, and produce abundant spores57. According to this concept, the inoculum fungus R. irregularis (R-10) might have typical ruderal traits; it colonizes roots in early growth stages as observed in this study, produces abundant spores3, and is well adapted to agricultural ecosystems (Peyret-Guzzon et al.58), which would be the essential traits for agriculture application. On the other hand, given that the indigenous fungi that competed with R-10 fungus were those that showed a higher value of commonness, their life history strategy is the same as that of R-10 fungus; they might also be ruderal. It is considered that these indigenous ruderals might largely proliferate during the cropping of palisade grass in the previous year 2014, resulted in the increases in MPN in T3_PG, because our protocol for the assessment of MPN would detect mainly the propagule that is capable of colonizing rapidly (i.e. within 26 days, Supplementary Methods S1). Terefore, population size of indigenous ruderal fungi, which could be estimated by MPN, is likely to be a key to predict the level of the colonization of inoculum fungus in the feld. Introduction of excess AM fungal propagule to the soil with high potential of indigenous AM fungi may decrease plant growth responses to the fungi. Janoušková et al.59 demonstrated that AM fungal inoculation, particularly at high rates of infective propagule, decreased host growth responses to the inoculation in the presence of a preestablished synthetic community in a model experiment. Tey suggested that excess propagule might enhance competition among the fungi for host resources, which consequently decreased the beneft of AM symbiosis. Our results that grain yield was decreased by the inoculation in T3_PG in which propagule density was higher than the others support their fndings at the feld level. In this study, a new primer FLd3 was frst designed as a mixture of the four diferent oligo DNAs in the conserved region between the D1 and D2 in the LSU rDNA. It was unexpected, however, that the OTU compositions obtained with each of the oligo DNAs were highly similar to each other. Basically, FLd3 was designed by taking into account the sequence variations in the region within the Glomeromycotina, because sequence mismatches generally bias the compositions in amplicon-based studies60. It seems likely that the annealing temperature (48 °C) in the thermal cycling program, which was determined according to the melting temperature (Tm) of the reverse primer FLR2, was actually 6–8 °C lower than the Tm of the four oligo DNAs and thus might allow them to anneal to the priming site even in the presence of one or two mismatches/gaps. Te fact that the OTU composition obtained with the eukaryote-universal primer LR1 was also highly similar to that obtained with FLd3 further indicates the validity of this primer; it is capable of amplifying a wide range of glomeromycotinan fungi with a minimum bias as confrmed with LR1 primer11,28,30. Conclusion Our modeling demonstrated that the dominance of inoculum fungus in the fowering stages is a necessary condition to obtain positive yield responses to the inoculation, which would be largely determined by agricultural practices/management, e.g., via altering the propagule density of indigenous AM fungi. In addition to agricultural practices/management, plant genotypes, which were not tested in this study, also critically afect growth responses to AM fungal inoculation (e.g., Sawers et al.48) and thus could be an important factor. More feld trials covering a wide range of plant cultivars in diverse environments are necessary to establish a more practical prediction model. Te analysis on niche competition between the introduced and indigenous fungi suggests that ruderal fungi play a key role in conventional (intensive) agriculture, which raises a question of how signifcant the ruderal traits are in less-intensive agriculture, such as no-till management and organic agriculture. Verbruggen et al.10 observed that species richness of AM fungi declines progressively with increasing management intensity. Tis observation leads to the idea that the fungi in less-intensive agricultural felds would be more diverse not only taxonomically but also functionally; for example, the ‘competitor’ fungi that proliferate mainly through hyphal networks54 may dominate in no (or reduced)-till felds instead of ruderal fungi. It is of interest to characterize AM fungal communities in less intensive agricultural felds through introduction of a ruderal fungal inoculum. References 1. Smith, S. E. & Read, D. J. Mycorrhizal Symbiosis, 3rd edn (Academic Press, 2008). 2. Pellegrino, E. et al. Establishment, persistence and efectiveness of arbuscular mycorrhizal fungal inoculants in the feld revealed using molecular genetic tracing and measurement of yield components. New Phytol. 194, 810–822, https://doi.org/10.1111/ j.1469-8137.2012.04090.x (2012). 3. Ceballos, I. et al. 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Efects of inoculum additions in the presence of a preestablished arbuscular mycorrhizal fungal community. Appl Environ Microbiol 79, 6507–6515, https://doi.org/10.1128/aem.02135-13 (2013). 60. Sipos, R. et al. Efect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis. FEMS Microbiol Ecol 60, 341–350, https://doi.org/10.1111/j.1574-6941.2007.00283.x (2007). Acknowledgements We are grateful to Drs. M. Hayashi, T. Karasawa, and S. Deguchi in National Agriculture and Food Research Organization (NARO), T. Sato in Akita Prefectural University, and T. Yagi in Hokkaido Research Organization for providing the test samples. We are grateful to the feld management staf of NARO for their technical assistance in the feld trials. Tis work was supported by ACCEL (JPMJAC1403) form Japan Science and Technology Agency. Author Contributions R.N., S.Y., T.K., K.A. and T.E. planned and designed the research. R.N., S.S., H.H., S.Y. and T.E. performed the MiSeq sequencing and developed the pipeline for community analysis. T.K. and K.A. performed the feld experiments. R.N., T.S., K.T. and T.E. defned the DNA tracking marker for the inoculum fungus. R.N., T.K. and T.E. analyzed the data and wrote the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-25701-4. Competing Interests: Te authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Scientific REPOrtS | (2018)8:7419 | DOI:10.1038/s41598-018-25701-4 11 The ISME Journal (2015) 9, 1053–1061 OPEN & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej PERSPECTIVE The role of community and population ecology in applying mycorrhizal fungi for improved food security

Alia Rodriguez1,3 and Ian R Sanders2,3 1Soil Microbiology, Faculty of Science, National University of Colombia, Ciudad Universitaria, Bogota´, Colombia and 2Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

The global human population is expected to reach B9 billion by 2050. Feeding this many people represents a major challenge requiring global crop yield increases of up to 100%. Microbial symbionts of plants such as arbuscular mycorrhizal fungi (AMF) represent a huge, but unrealized resource for improving yields of globally important crops, especially in the tropics. We argue that the application of AMF in agriculture is too simplistic and ignores basic ecological principals. To achieve this challenge, a community and population ecology approach can contribute greatly. First, ecologists could significantly improve our understanding of the determinants of the survival of introduced AMF, the role of adaptability and intraspecific diversity of AMF and whether inoculation has a direct or indirect effect on plant production. Second, we call for extensive metagenomics as well as population genomics studies that are crucial to assess the environmental impact that introduction of non-local AMF may have on native AMF communities and populations. Finally, we plead for an ecologically sound use of AMF in efforts to increase food security at a global scale in a sustainable manner. The ISME Journal (2015) 9, 1053–1061; doi:10.1038/ismej.2014.207; published online 31 October 2014

Introduction mycorrhizal fungi (AMF) are the two key groups of soil microorganisms with a potential for improving The problem of producing enough food to feed the nitrogen and phosphorus acquisition by crops, planet, and the need for increased food security, has respectively (Cakmak, 2002; Conniff, 2013; Reid become all too apparent in recent years. With a and Greene, 2013). Although significant advances global human population exceeding 7 billion, and are being made to get effective nitrogen-fixing estimates of over 9 billion by 2050, global food bacteria to farmers in developing countries (i.e. the production will have to be greatly increased (FAO, Bill and Melinda Gates Foundation-funded N2Africa; 2006; Godfray et al., 2010), especially in tropical and http://www.n2africa.org/), the application of AMF has subtropical regions where population growth rate is not been well adopted or lived up to the promises, increasing faster than in the rest of the world despite its enormous potential (Box 1). (The Future of Farming, 2011). Such yield increases In this perspective article, we consider how exceed the current global capacity to produce food appropriate application of AMF could improve food by prevalent farming practices, highlighting the security, by increasing the overall yield of important need to develop new technologies and better apply staple crops irrespective of the mechanism by which long-known technologies, such as growth-promoting it occurs (e.g. improved phosphate acquisition, microbial symbionts of plants, in a more efficacious improved drought or disease resistance). By food manner (Bennett et al., 2013). security crops, we mean those crops that are grown Poor soil fertility, particularly the availability of because they can feed a significantly large number of nitrogen and phosphorus, is the most limiting to people and because their yields fluctuate little increasing crop yields (Tilman et al., 2002). Conse- during periods of major climatic perturbation. We quently, nitrogen-fixing bacteria and arbuscular are not intending to review here the mechanisms by which the AMF symbiosis could give a crop species Correspondence: IR Sanders, Department of Ecology and Evolu- more food security-enhancing properties such as tion, University of Lausanne, Biophore Building, 1015 Lausanne, increased drought resistance, disease resistance or Switzerland. by other systemic effects revealed by recent tran- E-mail: [email protected] 3These authors contributed equally to this work. scriptome profiling studies, although we recognize Received 5 May 2014; revised 18 September 2014; accepted 24 that these aspects are extremely important (e.g. see September 2014; published online 31 October 2014 Salvioli and Bonfante, 2013; Zouari et al., 2014). The need for ecology in applying mycorrhizal fungi A Rodriguez and IR Sanders 1054 Here, we outline two major challenges to Box 1 Enormous potential for mycorrhizal fungi (i) effectively and (ii) safely using AMF to to improve food security in the tropics significantly improve production of important food security crops, especially in the tropics. We The potential of AMF to help increase global food believe that both these challenges can only be security lies in the fact that all globally important resolved by the adoption of community food crops naturally form this symbiosis and the and population ecology approaches. We propose fungi help plants more efficiently obtain phos- possible ways to overcome past hurdles in apply- phate from the soil (Smith and Read, 2008). ing AMF inoculation technology by respecting Stocks of phosphate fertilizer are rapidly being the general community and population ecological depleted (Gross, 2010). There is a simultaneous principles. These perspectives are based on increase in demand for phosphate to help feed the recent ecological, community and population growing population (Gilbert, 2009). These two genetic insights, coupled with recent techno- combined factors represent a major threat to logical advances that allow us to better understand global food security; a threat that can potentially the ecology of these important fungi. We focus be reduced by better phosphate acquisition particularly on the tropics because most tropical through the AM symbiosis. The potential of soils are acidic and very nutrient poor, especially AMF to contribute to improved crop yields has in bioavailable phosphate. It is in these soils been known for decades. However, despite where we consider that AMF could make the an extremely strong research focus on this largest contribution, but have been most symbiosis,* there are remarkably few published overlooked. studies demonstrating that large-scale inoculation of globally important crops, in an agricultural situation, results in significant increases in food production. The reasons for this are (taken from Challenge 1: AMF are everywhere! A major Ceballos et al., 2013): ecological challenge to effectively using AMF in agriculture (1) Mycorrhizal fungi are already present in all We argue that there is a major ecological challenge to agricultural soils. Effective AMF inoculation effectively using AMF to increase crop production, requires yield increases over those attained where agronomists cannot afford to ignore microbial in the non-inoculated crop. community and population ecology. Thousands of (2) Most AMF field trials have been conducted published studies conducted in pots with sterile in temperate agroecosystems where nutrient soil, where inoculated versus non-inoculated plants bioavailability is high compared with most were compared, form the basis of the well-known tropical soils, thus reducing the possibility potential of AMF to improve plant growth. However, of an AMF effect. In contrast, most tropical all soils in which crops are cultivated already soils have low bioavailable phosphate con- contain diverse communities of AMF and all tent and high phosphate retention capacities globally important food crops naturally become (Friesen et al., 1997). It is exactly in these colonized by AMF independently from inoculation. soils where the application of AMF has the Consequently, expecting that introducing AMF to an strongest potential to increase food produc- already established AMF community will lead tion and reduce the need to apply phosphate to consistent increases in crop yield is utterly fertilizers, but has been the most neglected. simplistic. We already know that microbial commu- (3) AMF inoculum can only be produced with nity structure and diversity can greatly affect plant plants. Culturing AMF traditionally requires productivity and interactions among these micro- large-scale production of plants, from which organisms can even allow persistence of less the AMF inoculum can be harvested. Often beneficial microbes (Rosendahl, 2008; Bever et al., this inoculum is simply soil containing 2013; Hart et al., 2013). It is unsurprising, therefore, AMF. It is difficult to ensure consistent that there are very few published field trials with inoculum quality (Gianinazzi and Vosa´tka, AMF that have given consistently promising results 2004), impossible to ensure that the soil/ with a major food crop that is responsible for feeding carrier is free from other microorganisms and a significant proportion of the global human popula- the weight can make transport costs prohibi- tion (perhaps with one exception; Box 2). There are tively high in developing countries. additional reasons for poor adoption of mycorrhizal technology (Box 1). We propose that a better under- *A survey of the ISI Web of Science database standing of the ecology and population biology of revealed 1441 publications on the mycor- AMF, and how introduced AMF interact with rhizal symbiosis in 2013, of which we existing AMF communities and populations, is assume at least half are probably on the essential to developing effective AMF inocula for arbuscular mycorrhizal symbiosis. increasing yields of the globally important crops, namely rice, wheat, cassava, maize and potatoes.

The ISME Journal The need for ecology in applying mycorrhizal fungi A Rodriguez and IR Sanders 1055 We identify four areas where ecologists could Box 2 Cassava and AMF revisited contribute significantly to a more effective use of AMF: The response of most globally important crops to inoculation with AMF has been unpredictable Understanding the survival and colonizing ability and far from spectacular except for cassava of introduced AMF in the presence of an existing (Manihot esculenta Crantz). Cassava is one of the most important tropical crops providing AMF community. Understanding the adaptability of AMF to envir- almost a billion people with a third of their daily onmental conditions that the fungus has not caloric intake in 105 countries (FAO, 2010). Cassava cropping is encouraged by the FAO to previously experienced. The importance of within-AMF species genetic improve food security because yields of cassava variation and how it affects plant growth. fluctuate much less compared with cereals during The need to identify whether the effect on crop yield periods of climatic perturbation (FAO, 2005). of introduced AMF is direct or indirect, through changes to the pre-existing AMF community. Cassava appears to be strongly dependent on mycorrhizal fungi for growth and nutrition (Howeler and Sieverding, 1983; Sieverding and Do introduced AMF actually establish? Howeler, 1985). In a series of pioneering field experiments, E Sieverding and colleagues at the Understanding how introduced AMF interact and International Centre for Tropical Agriculture in coexist with the local AMF community and whether Colombia conducted many trials inoculating this directly leads to changes in crop production is cassava with AMF (their work is summarized in key to a successful application of AMF in agricul- Sieverding, 1991). They demonstrated that large, ture. Remarkably, few field studies have directly consistent yield increases could be achieved by linked yield increases with successful colonization inoculating cassava with AMF, in the presence of by an introduced AMF. One field study in which an existing AMF community in Colombian soils. yield increases were observed, with parallel Also, inoculation in one crop cycle had a increases in AMF colonization, revealed that one continued effect in subsequent crop cycles, out of two introduced AMF inoculants may have indicating either a persistence of the introduced successfully established and persisted 2 years AMF or a beneficial durable change to the following inoculation (Pellegrino et al., 2012). The existing AMF community. pending question is whether that particular isolate was directly responsible for the increase in biomass The use of AMF in cassava cropping has not of the target plant or even of the higher mycorrhizal been adopted because without a mass inoculum colonization observed in those roots. Nowadays, production system, the application was imprac- high-throughput sequencing offers a more powerful, tical. Currently, in vitro-grown R. irregularis can sensitive and also quantitative technique to track the be mass produced in a sterile carrier—ensuring fate of the introduced AMF, both temporally and inoculum quality and an easy to transport spatially and also determine whether the establish- product. Recently, we have successfully used ment of the AMF is influenced by the native AMF in vitro-produced R. irregularis to significantly community (Figure 1a). It remains to be seen increase cassava yields in Colombia (Ceballos whether effective inoculation of crops with AMF et al., 2013). The application of the fungi will lead to significant levels of colonization by the requires no skill and it significantly increased introduced fungus, but as we explain below, this cassava yields by B20% (with a maximal yield may not necessarily be important if the effects are of 45 t ha À 1). The effects were observed in two indirect via the resident AMF community. sites where the soils, climate and cassava variety were different. Do AMF inoculants have to be preadapted We believe that because high-quality mass- to a particular soil or crop species? produced and easily transportable AMF inocu- Very little is known about adaptation of AMF to lum is available, the largely forgotten work, environmental conditions (Johnson et al., 2013). pioneered by Sieverding and Howeler (1985), A common assumption for field applications is that needs to be revisited as it offers an important to be effective, the fungus has to be adapted to a and unique opportunity where the food produc- given soil type or crop. Indeed, as high nutrient tion and security of a globally important crop levels can reduce colonization by AMF, it could could be significantly increased through the be more difficult for AMF to establish in more application of AMF. nutrient-rich soils. However, some AMF species appear to have an extremely large geographic range, suggesting a lack of specialization to certain environments (i.e. Funneliformis mosseae and

The ISME Journal The need for ecology in applying mycorrhizal fungi A Rodriguez and IR Sanders 1056

AMF community effects AMF population effects

*Introduced AMF exchanges 1 Local AMF community *Introduced AMF estasblished but 1 Local AMF population local AMF community unaltered DNA with local AMF 4 4 l e

F v

o M t n Introduction of an AMF strain A n l Introduction of an F o e t a r of a different species M e c AMF inoculum e f A o s f l (strain of i i d r d e h t s y 2 c i indigenous species) l e l v u w i a d n n g o 2 o r o e i *Establishment depends on t i t t g n c i c n n local AMFcommunity composition f a a r h fu o e c t t e c x r in e fe a f s ic t e it *Introducing AMF results in t a t e h c h n t *Introduced AMF alters e g 3 presence of novel alleles in e F 3 ir u G M community composition, diversity D ro A h the population n t tio or structure r r la o o pu ct po ire F he ct r d AM in t ffe ithe al pread al e op e loc Alleles may s tion 5 POTENTIAL the cr n POTENTIAL unc Effect on ge o g the f *Introduced AMF FUNCTIONAL OUTCOME chan FUNCTIONAL OUTCOME changin indirect through (or genetically novel descendents) spreads and becomes invasive Figure 1 Potential effects of inoculating the tropical crop cassava with an AMF. (a) Effects and functional outcomes that are potentially mediated by the presence of an AMF community. (b) Effects and potential functional outcomes when a population of the same AMF species already exists in the field. Asterisk denotes hypotheses that can only be verified by experimental investigation as proposed in this article.

Rhizophagus irregularis in Rosendahl et al., 2009 AMF species versus within-species and Box 3). In fact, some field studies successfully diversity used in vitro-grown R. irregularis from arid Spanish soil in extremely nutrient-poor tropical acidic soils AMF species richness is a major contributor to and with a plant (cassava) that the fungus had not maintaining plant species diversity and ecosystem previously experienced (Ceballos et al., 2013). functioning (van der Heijden et al., 1998a). Many The question of environmental adaptation in field investigations have also demonstrated that AMF has only been tangentially approached crops respond differently to inoculation with differ- (Johnson et al., 2010). Johnson et al. (2010) ent AMF species. Most of these studies demonstrat- provided the first evidence, in natural ecosystems, ing differential effects of AMF species on plants of possible local AMF community adaptation to have used one individual AMF isolate as a repre- soil mineral nutrient levels. However, recent sentative of the species. However, one study shows laboratory evidence suggests a genetic mechanism that larger variation in plant biomass can be induced that could allow for the rapid adaptation in AMF to by randomly choosing two different isolates of the a change of environment (Angelard et al., 2014). same species, rather than inoculating with two Strains of R. irregularis that had been maintained different species (Munkvold et al., 2004). Furthermore, for B12 years on carrot roots exhibited rapid variation in plant growth caused by different AMF genetic change following a shift to potato and individuals from one population has been shown to showed a very large range of phenotypic responses have a genetic basis, highlighting genetic variation in to a change of host. Thus, to understand whether AMF populations as a major source of variation (Koch AMF need to be adapted to a given environment, et al., 2006). Furthermore, a fivefold difference in the we also need to understand the mechanisms by growth of rice was observed by inoculating with whichAMFcanbecomeadaptedtoanew genetically different R. irregularis isolates (Angelard environment. et al., 2010). This calls for further studies on the Finally, we see little available experimental data functional implication of genetic diversity in AMF on whether AMF that have evolved in a given populations as it may be possible to breed and select environment will be more effective in that environ- more effective AMF for crop plants. ment. Thus, we believe that in order to more We also stress that genetic variation in crop effectively use AMF, it is necessary to understand species also needs to be considered as AMF the role that selective adaptation has in the effec- genotype-by-crop genotype interactions may exist tiveness of AMF in given soils or crops. where crop varieties respond differently to a range

The ISME Journal The need for ecology in applying mycorrhizal fungi A Rodriguez and IR Sanders 1057 Direct versus indirect effects of AMF Box 3 The case of Rhizophagus irregularis as a inoculation commercial inoculant Agricultural soils have been shown to harbour The AMF species Rhizophagus irregularis has diverse AMF communities (Smith and Read, 2008). become the model AMF species because it is Ecological studies have clearly shown that different easily cultured in an in vitro system (Be´card and species of AMF induce different effects on plant Fortin, 1988) and its genome has recently been growth (van der Heijden et al., 1998b). Thus, it sequenced (Tisserant et al., 2013). In vitro appears likely that alterations to the AMF commu- cultivation makes it possible to obtain fungal nity could potentially alter the yield of a crop DNA that is free of DNA of other organisms (Koch without necessarily changing overall colonization et al., 2004). The fungus has a worldwide levels caused by adding an additional AMF species. distribution. An unpublished survey revealed Introducing a non-indigenous AMF is a biotic that B300 different isolates of this fungal species disturbance that could alter resident AMF commu- probably exist in laboratories worldwide (R nity structure (Figure 1a). There is no information, Savary, personal communication). This means however, on whether AMF inoculation that results that it is ideal for studies of AMF population in improved crop yield is actually because of a direct genetics (Koch et al., 2004; Bo¨rstler et al., 2008; effect of the introduced AMF on the plant or indirectly Croll et al., 2009). through a change in the local AMF community (Figure 1a). It is, therefore, highly pertinent to under- A big advantage of R. irregularis is that it can stand the mechanisms that govern AMF community readily be put into in vitro culture and be composition in response to application of non- efficiently mass produced (Be´card and Fortin, indigenous AMF. This is a key ecological issue in 1988). We consider this fungus to be of utmost need of thorough consideration for an ecologically importance in the future applications of AMF to sustainable use of AMF (see Challenge 2). Further- improve food security because of: (1) its world- more, variable effects on crop yield of AMF applica- wide distribution; (2) its high genetic variability tion, using the same inoculum in different places, and variation in effects on plant growth; (3) the might be determined by their indirect effect on local ability to carry out population genomics studies AMF communities, which might differ in these places. and marker development on this fungus; and (4) At present, there is almost no information about the ability to mass produce this fungus efficiently what determines the community composition of and its effects on yields of a globally important AMF in a given environment and, indeed, whether crop. there are abiotic or biotic factors that allow us to predict which AMF taxa will form a given assem- Critics may argue that it would be unwise to blage in a given locality. Without understanding of promote the use of one AMF species. However, at such basic ecological patterns, which we largely present, we see this as the most pragmatic option take for granted in plant and animal assemblages, it because of the ease to culture this fungus in vitro is impossible to even start predicting how a given (unlike most other AMF species). Nevertheless, AMF community might react to the introduction of we emphasize that the appropriateness of using an additional AMF inoculum. One way to address one species is entirely dependent on the niche this is if community ecologists describe AMF range of the fungus, the amount of global genetic community structure over a wide variety of variability in this species and how much this well-described agroecosystems to identify the variability contributes to variation in plant environmental variables that determine AMF growth, all of which can only be known by community assemblages. With this knowledge, undertaking the population genetics studies pro- for a large number of sites, it would then be posed here. However, it should be noted that for possible to select sites with given environments the last 8 millennia, the global human population and AMF assemblages and perform experiments to has received the majority of its nourishment and observebothcropresponsesandAMFcommunity daily calories from a only small handful of crop responses to inoculation with one uniform species and low species number has not been a inoculum. With modern molecular techniques, major problem limiting food production, so this this very large experiment is technically possible may be the case for AMF species as well. and we see this as critical for predicting how a given AMF inoculum will affect already existing AMF assemblages. of different AMF genotypes. Different plant geno- Challenge 2: Ecological impact of types are known to respond differently to AMF introducing non-native AMF inoculation, but so far the interaction has not been investigated in sufficient detail (Hetrick et al., 1995; The second major challenge we highlight is the need An et al., 2010). to understand the ecological impact of introducing

The ISME Journal The need for ecology in applying mycorrhizal fungi A Rodriguez and IR Sanders 1058 AMF into soils in which they previously did not community. Although a decrease in AMF diversity occur. This was first addressed in a landmark is likely to be considered as a negative environ- publication that called for caution in global dis- mental impact, we still do not understand which persal of AMF inoculants and stressed the need for aspects of AMF diversity (richness, evenness) environmental impact studies (Schwartz et al., favour crop growth. The experimental approach 2006). Recent development of new techniques suggested above (end of Challenge 1) for investi- means this can now be assessed and, owing to new gating direct versus indirect effects should help to findings, additional factors need to be taken into unravel this problem. consideration. Without conducting the research that we highlight below, it is impossible to assess the safety and dangers associated with introducing Persistence and invasiveness of introduced non-native AMF. The recent developments in the AMF: a population genomics approach analysis of microbial communities, populations and AMF inoculants are difficult to trace in field genomes make it feasible to address the following experiments. Therefore, it has been difficult, in questions: practice, to measure either the persistence or the invasiveness of an introduced AMF. Consequently, Does an introduced AMF alter the composition there is no data available on this topic (Figure 1b). and/or structure of the naturally occurring AMF A further practical problem is that the same fungal community? species may already be present in the native AMF Do introduced AMF persist and spread in the community, which means that a large number of environment? molecular markers used for tracing the fungus may If the introduced AMF species already occurs already exist at the field site. However, we propose locally, are we introducing new genetic material that it is potentially possible to study invasiveness (new alleles) into an existing AMF population? and persistence of one particular AMF species, Will the introduced fungus undergo genetic namely R. irregularis (Box 3), by adopting a popula- exchange with the local AMF population and tion genomics approach, even though this fungus is what are the functional consequences? already present in many agricultural fields. With the recent publication of the reference genome of R. irregularis isolate DAOM 197198 (Tisserant et al., 2013), it is now easier to undertake Effects on AMF communities of partial genome sequencing of multiple R. irregularis introducing non-native AMF isolates and obtain a set of specific polymorphic markers to discriminate introduced R. irregularis An obvious question is whether the introduction strains from local ones. The technique known as of a non-native AMF significantly alters either the random amplified polymorphic DNA sequencing is diversity or the composition of the existing AMF a powerful, and also an extremely reliable, high- community. A number of studies have used throughput method for identifying large numbers of pyrosequencing of rDNA amplicons from roots or polymorphisms across genomes of different indivi- soil, giving the first picture of AMF diversity in duals (Baird et al., 2008). Potential marker identifi- communities (Lindahl et al., 2013; O¨ pik et al., cation would then be based on polymorphisms 2013). However, the technology needs to be between the introduced R. irregularis strain and suitable for large sample numbers and high polymorphisms of a large number of R. irregularis replication, as well as for having the appropriate isolates whose genomes were sequenced. bioinformatics tools to handle very large data sets (Pagni et al., 2013). At the time of writing, Illumina MiSeq technology produces B30 million Introduction of non-native genetic paired-end 300 bp reads, which means that a material into existing AMF populations: B550 bp concatenated amplicon sequence should the need for population genomics give a very large number of informative reads, making it possible to work with considerable Introducing non-native AMF at sites where the same sequence coverage per amplified sample (Smith AMF species occurs could result in the introduction and Peay, 2014). Pooling of barcoded amplicons of new or very different genetic material into from large numbers of samples into single libraries existing fungal populations even though no new will allow large-scale studies on AMF commu- species is introduced. However, based on our nities from inoculated and non-inoculated soils. knowledge of AMF genetic variation, these newly New more robust clustering algorithms are introduced individuals could potentially be geneti- also being developed, such as DBC454, that are cally very different from those that are already efficient for the processing of large data sets (Pagni present. This can be considered highly relevant et al., 2013). because we already know that genetically different A much greater challenge is the ecological isolates of one AMF species can cause large interpretation of any changes to the local AMF differences in plant growth, even if they originate

The ISME Journal The need for ecology in applying mycorrhizal fungi A Rodriguez and IR Sanders 1059 from the same population (Koch et al., 2006; field and its consequences to be technically challen- Angelard et al., 2010). ging, but feasible with current knowledge of the To know the likelihood of adding new alleles into R. irregularis genome and powerful high-throughput indigenous AMF populations, we believe that a polymorphism detection tools. detailed study of global population genomics of an AMF species is urgently needed. The fungus R. irregularis is an ideal candidate (Box 3). Some Conclusions information on the population genetics of this We conclude that ecological approaches at commu- fungus already exists and is intriguing. In a study nity and population scales, made possible by of a Swiss R. irregularis population, where isolates metagenomics and population genomics tools, can originated from 1 small field (100 Â 100 m2), genetic pave the way to a better informed agronomic differences among isolates were very large (Koch utilization of AMF. As we point out, modern et al., 2004; Croll et al., 2008). However, there were molecular techniques are already available for these very few alleles in a Canadian isolate (DAOM studies and it is the responsibility of microbial 197198) that did not already exist in the Swiss ecologists and agronomists to take up these chal- population. This leads to the question whether lenges, as their contribution could help lead to R. irregularis populations might be locally extremely practical solutions to the problem of producing more diverse, but globally not much richer. If true, it food in a sustainable manner. would mean that introducing inoculants of R. irregularis into soils from which the inoculant does not originate may pose little risk for the Conflict of Interest introduction of significant amounts of new genetic material (Figure 1b). However, only thorough The authors declare no conflict of interest. population genomics studies using high-throughput polymorphism detection approaches (such as Acknowledgements random amplified polymorphic DNA sequencing) on globally distributed R. irregularis isolates can IRS and AR gratefully acknowledge the financial support help answer this question. from a Swiss National Science Foundation Joint Research Project (project number IZ70Z0_131311/1) that allowed the development of these ideas and constructive comments from three anonymous reviewers. Genetic exchange between introduced AMF, the existing population and its consequences It has been demonstrated that the fungus References R. irregularis anastomoses with genetically different individuals of the same species, giving rise to An GH, Kobayashi S, Enoki H, Sonobe K, Muraki M, genetically novel progeny, whose effects on plant Karasawa T et al. (2010). How does arbuscular growth differ from those of the parents (Croll et al., mycorrhizal colonization vary with host genotype? 2009; Angelard et al., 2010; Colard et al., 2011). An example based on maize (Zea mays) germplasms. Thus, anastomosis between an introduced AMF and Plant Soil 327: 441–453. local AMF of the same species would potentially Angelard C, Colard A, Niculita-Hirzel H, Croll D, allow non-native alleles to mix with the local Sanders IR. (2010). Segregation in a mycorrhizal fungus alters rice growth and symbiosis-specific gene population. This has the potential to change how transcription. 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The ISME Journal The need for ecology in applying mycorrhizal fungi A Rodriguez and IR Sanders 1061 Sieverding E, Howeler RH. (1985). Influence of van der Heijden MGA, Klironomos JN, Ursic M, species of VA mycorrhizal fungi on cassava yield Moutoglis P, Streitwolf-Engel R, Boller T et al. response to phosphorus fertilization. Plant Soil 88: (1998b). Mycorrhizal fungal diversity determines 213–221. plant biodiversity, ecosystem variability and produc- Smith DP, Peay KG. (2014). Sequence depth, not PCR tivity. Nature 396: 69–72. replication, improves ecological inference from next Zouari I, Salvioli A, Chialva M, Novero M, Miozzi L, generation DNA sequencing. PLoS One 9: e90234. Tenore GC et al. (2014). From root to fruit: RNA-Seq Smith SE, Read DJ. (2008). The Mycorrhizal Symbiosis. analysis shows that arbuscular mycorrhizal symbiosis Academic Press: San Diego, CA, USA. may affect tomato fruit metabolism. BMC Genom The Future of Farming (2011). Final Project Report. The 15: 221. Government Office for Science: London, UK, p 211. Tilman D, Cassman KG, Matson PA, Naylor R, Polasky S. This work is licensed under a Creative (2002). Agricultural sustainability and intensive Commons Attribution 3.0 Unported production practices. Nature 418: 671–677. License. The images or other third party material in Tisserant E, Malbreil M, Kuo A, Kohler A, Symeonidi A, this article are included in the article’s Creative Balestrini R et al. (2013). Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant Commons license, unless indicated otherwise in the symbiosis. Proc Natl Acad Sci USA 110: 20117–20122. credit line; if the material is not included under the van der Heijden MGA, Boller T, Wiemken A, Sanders IR. Creative Commons license, users will need to obtain (1998a). Different arbuscular mycorrhizal fungal permission from the license holder to reproduce the species are potential determinants of plant community material. To view a copy of this license, visit http:// structure. Ecology 79: 2082–2091. creativecommons.org/licenses/by/3.0/

The ISME Journal Ecology Letters, (2006) 9: 501–515 doi: 10.1111/j.1461-0248.2006.00910.x IDEAS AND PERSPECTIVES The promise and the potential consequences of the global transport of mycorrhizal fungal inoculum

Abstract Mark W. Schwartz,1* Jason D. Advances in ecology during the past decade have led to a much more detailed Hoeksema,2 Catherine A. understanding of the potential negative consequences of speciesÕ introductions. Gehring,3 Nancy C. Johnson,3 Moreover, recent studies of mycorrhizal symbionts have led to an increased knowledge 4 John N. Klironomos, Lynette K. of the potential utility of fungal inoculations in agricultural, horticultural and ecological 5 6 Abbott and Anne Pringle management. The intentional movement of mycorrhizal fungal species is growing, but 1 Department of Environmental the concomitant potential for negative ecological consequences of invasions by Science & Policy, University of mycorrhizal fungi is poorly understood. We assess the degree to which introductions of California, Davis, CA, USA 2 mycorrhizal fungi may lead to unintended negative, and potentially costly, consequences. Department of Ecology and Evolutionary Biology, University Our purpose is to make recommendations regarding appropriate management guidelines of California, Santa Cruz, CA, and highlight top priority research needs. Given the difﬁculty in discerning invasive USA species problems associated with mycorrhizal inoculations, we recommend the 3Department of Biological and following. First, careful assessment documenting the need for inoculation, and the Environmental Sciences, likelihood of success, should be conducted prior to inoculation because inoculations are Northern Arizona University, not universally beneﬁcial. Second, invasive species problems are costly and often Flagstaff, AZ, USA impossible to control by the time they are recognized. We recommend using local 4 Department of Integrative inoculum sources whenever possible. Third, non-sterile cultures of inoculum can result Biology, University of Guelph, in the movement of saprobes and pathogens as well as mutualists. We recommend using Guelph, Ontario, Canada material that has been produced through sterile culture when local inoculum is not 5School of Earth and available. Finally, life-history characteristics of inoculated fungi may provide general Geographical Sciences, The guidelines relative to the likelihood of establishment and spread. We recommend that, University of Western Australia, Perth, WA, Australia when using non-local fungi, managers choose fungal taxa that carry life-history traits that 6Organismic and Evolutionary may minimize the likelihood of deleterious invasive species problems. Additional Biology, Harvard University, research is needed on the potential of mycorrhizal fungi to spread to non-target areas Cambridge, MA, USA and cause ecological damage. *Correspondence: E-mail: [email protected] Keywords Agriculture, dispersal, horticulture, inoculum, invasive species, mutualism, mycorrhizae, restoration, symbiosis.

Ecology Letters (2006) 9: 501–515

(McKinney & Lockwood 1999), and costly management of INTRODUCTION noxious invaders (US Congress Office of Technology As humans continue to intentionally, and unintentionally, Assessment 1993; Pimentel et al. 2000, 2005). As a response move species around the planet, it is increasingly important to these negative ecological consequences, databases (e.g. to understand both the benefits and costs of these actions. Global Invasive Species Database), councils (e.g. US Understanding the potentially large consequences of glob- National Invasive Species Council; Clinton 1999) and alization of species distributions has become a major focus numerous local laws and policies (Miller & Fabian 2004) of ecological studies during recent decades. This globaliza- have been created to slow the wave of invasion. Never- tion of biota has resulted in: (a) ecological degradation and theless, there have also been enormous economic benefits degraded ecosystem services (Mack et al. 2000); (b) biodi- associated with intentional species movement. For example, versity losses; and (c) increased biotic homogenization virtually all of agricultural production is a product of species

Ó 2006 Blackwell Publishing Ltd/CNRS 502 M. W. Schwartz et al. in non-native habitats. Although it is not often discussed in of at least three cryptic species including a Eurasian lineage, the current ecological literature on invasive species, the a Eurasian sub-alpine lineage, and a North American lineage societal benefits and economic gains as a consequence of (Oda et al. 2004). Thus, A. muscaria appears to be a moving biota in an effort to support human societies is widespread distributed morphospecies with cryptic genetic considerable. species, at least one of which has been introduced to a novel Within this context, the rate and volume of the intentional continent. An isolate from New Zealand groups with movement of non-indigenous mycorrhizal fungi is increasing Japanese A. muscaria, suggesting an Asian origin for as a consequence of the promise of harnessing beneficial soil A. muscariaÕs invasion (Oda et al. 2004). The example organisms for improved agriculture (Gianinazzi et al. 2002), illustrates two points. First, cryptic species have the horticulture (Azcon-Aguilar & Barea 1997), habitat restora- potential to invade each other’s ranges without detection tion (Miller & Jastrow 1992), bioremediation (Leyval et al. and so, potentially, displace native species. In this case, 2002), and forestry (Brundrett et al. 1996, Duponnois et al. A. muscariaÕs invasion of Australia is obvious because it is the 2005). The approach of this paper is to jointly examine our morphological species that has invaded. If a cryptic species understanding of mycorrhizal ecology along with general were to invade the range of another cryptic species (e.g. if a patterns of invasive species in order to produce a preliminary Eurasian lineage were to invade North America) then that assessment of the potential for costly unintentional outcomes invasion might well go unnoticed. Second, the impact of the of mycorrhizal inoculation. We then make recommendations invasive A. muscaria in Australia and New Zealand is to help minimize the risk of management mistakes using unknown, as we neither know if it is displacing native mycorrhizal fungi. Further, we suggest a research agenda to species nor if, through altered biogeochemistry, it has help fill existing knowledge gaps that make it difficult to ecosystem consequences. predict outcomes of inoculation with mycorrhizal fungi. Our Mycorrhizal fungi are generally considered mutualistic, goal is to highlight ways by which we might maximize and accordingly, there has been little concern over potential beneficial utility while minimizing risks associated with negative consequences of their introduction. Nevertheless, harmful species introductions. evidence is growing that mycorrhizal function can range To our knowledge, there are no documented cases where from mutualistic to parasitic (Johnson et al. 1997; Klirono- the intentional movement of mycorrhizal fungi has led mos 2003; Jones & Smith 2004) with host plant and edaphic directly to a widespread, persistent invasive species problem. conditions mediating their functioning. Within an old-field It is difficult to ascertain, however, whether this lack of plant community, Glomus etunicatum can stimulate the growth knowledge is because problems do not exist, or because they of certain plants but be detrimental to many others go undetected. The absence of documented problems from (Klironomos 2003). Enormous functional variability also introduced mycorrhizal fungi is in stark contrast to exists among species of ectomycorrhizal fungi in attributes problems caused by invasions of pathogenic fungi (e.g. such as the utilization of organic nitrogen sources (e.g. Dutch elm disease or chestnut blight). With respect to plant Abuzinadah and Read 1986) and tolerance of water stress disease issues, Anderson et al. (2004) reviewed the literature (e.g. Coleman et al. 1989). Thus, inoculation treatments must and surmised that the problem lies in a lack of detection. be supported by consideration of possible negative With the recent upsurge in the use of mycorrhizal inoculum, consequences along with the potential for benefit (Table 1). the potential for problem invasions may be increasing. Jonsson et al. (2001) observed threefold differences among There is a need to consider the possibility of both overt as mycorrhizal species in their ability to influence shoot well as subtle undesirable effects of the movement of biomass in Pinus sylvestris growing under the same environ- mycorrhizal fungi. Undesirable consequences of inoculation, mental conditions. In addition, mycorrhizal fungi can where they occur, are likely to go unnoticed because large- facilitate plant growth both through nutrient exchange as scale monitoring of the consequences of inoculation is rarely well as pathogen control (Whipps 2004). It is important to conducted. A case study illustrates some of these possible recognize this variability in function because mycorrhizal complexities associated with fungal invasions. The ectomy- fungi can influence crop yields, tree survival, plant commu- corrhizal fungus Amanita muscaria was introduced to nity structure and ecosystem properties (e.g. Johnson et al. Australia and New Zealand in the 19th century (Bougher 1992; van der Heijden et al. 1998; Jonsson et al. 2001). 1996; Orlovich & Cairney 2004) and frequently associates We focus this essay on arbuscular mycorrhizal (AM) and with trees endemic to its introduced habitats, e.g. Nothofagus ectomycorrhizal (EM) fungi, because they are the most spp. (Orlovich & Cairney 2004). The mushroom of widespread and economically important types of mycorrh- A. muscaria is typically bright red with white spots and it is izal fungi. These groups differ fundamentally in the easily identified by even the casual naturalist. The morphology of their root associations, as their names morphological species is found in Europe, Asia and North suggest. The AM fungi produce arbuscules within roots that America. However, molecular markers suggest the presence function as exchange surfaces with plants. In contrast, EM

Ó 2006 Blackwell Publishing Ltd/CNRS Global transport of mycorrhizal fungal inoculum 503

Table 1 Potential beneﬁcial and detrimental outcomes of inoculation with mycorrhizal fungi

Potential beneﬁcial consequences Potential detrimental consequences

Increased yields and survival of desirable plant species Decreased yields and survival of desirable plant species (Bethlenfalvay & Linderman 1992) (e.g. Hendrix et al. 1992) Reduced fitness of noxious invasive weeds (Johnson 1998) Increased fitness of noxious invasive weeds (e.g. Marler et al. 1999) Decreased uptake of toxic compounds (e.g. Rufyikiri et al. 2004) Increased uptake of toxic compounds (e.g. Killham & Firestone 1983) Reduced diversity of indigenous mycorrhizal fungi Improved soil aggregation and stability (Miller & Jastrow 2000) Enhanced carbon storage in soils (e.g. Hogberg & Hogberg 2002) Reduced carbon storage in soils (Chapela et al. 2001) fungi are named for their characteristic coat of hyphae that Production and application of mycorrhizal inoculum surrounds the external surface of roots. Although these groups differ in numerous other significant traits (Table 2), In 2001, there were more than 30 companies worldwide in both cases, the fungi receive carbon from the plants with marketing one or multiple products containing mycorrhizal which they associate in exchange for providing nutrients fungal inoculum (Gianinazzi & Vosatka 2004). These and/or pathogen protection to their host plants (Smith & products are marketed as plant growth promoters to be Read 1997). used in horticultural, agricultural, restoration and forestry

Table 2 Ecological attributes of AM and EM fungi.

AM fungi EM fungi

Number of described species < 200, but many undescribed species are known > 5000 (Molina et al. 1992) worldwide to exist (Bever et al. 2001; Clapp et al. 2002) Typical number of species < 50 (Bever et al. 2001; Clapp et al. 2002) Varies from < 10 to > 200 found locally (Horton & Bruns 2001) Host plants and habitats Herbaceous and woody plant hosts in many Woody plants in families Betulaceae, habitats from grasslands to forests, low to high Dipterocarpaceae, Fagaceae, Myrtaceae, and latitude, on c. 90% of all plant species Pinaceae, typically in forested habitats that (Smith & Read 1997) have lower plant diversity than AM-dominated habitats (Smith & Read 1997) Host specificity Generalists with respect to ability to form Varies widely: some are species-specific, some association, but strong intra- and interspecific genus-specific, some family-specific, and some variation in relative preference for and are broad generalists (Molina et al. 1992) performance with different host plant species is recently being discovered (Bever et al. 2001) Trophic capability Obligate biotrophs – cannot obtain carbon Some species have some saprobic capability without host plant association (Smith & Read 1997) (Smith & Read 1997) Spore size Small to large (10–1000 microns) Small (usually < 15 microns) (Smith & Read 1997) (Smith & Read 1997) Spore dispersal mode Animal vectors and/or physical soil movement Aerial dispersal, animal vectors, and/or physical (Allen 1991) soil movement (Allen 1991) Colonization speed Significant variation within and among species with respect to ability to quickly colonize host plants from spores (EM: e.g. Kennedy & Bruns 2005; AM: e.g. Hart & Reader 2002). Similar variation may exist for the ability to colonize by growing from one plant root system to another. Growth effect on host plants Both AM and EM fungi vary intra- and interspecifically with respect to impact on host plants, which can range from negative to neutral to positive (Smith & Smith 1996; Johnson et al. 1997; Klironomos 2003; Jones & Smith 2004) Competitive ability for host The sparse experimental evidence suggests that species vary widely in their ability to compete for access plant resources to host roots, and that competition is often asymmetric (AM fungi: Lopez-Aguillon & Mosse 1987; Hepper et al. 1988; Pearson et al. 1993 and 1994, Wilson & Trinick 1983; Wilson 1984; Sen et al. 1989; EM fungi: Wu et al. 1999; Landeweert et al. 2003; Kennedy & Bruns 2005; AM vs. EM: Chen et al. 2000)

Ó 2006 Blackwell Publishing Ltd/CNRS 504 M. W. Schwartz et al. applications. Typically, only a small number of different Linderman 1992), there are also many reports of neutral or mycorrhizal taxa are included in these products, the most even detrimental effects of mycorrhizal fungi on crops and common being Pisolithus tinctorius (an EM fungus) and trees in reforestation sites (e.g. Bledsoe et al. 1982; Teste Glomus intraradices (an AM fungus). Numerous methods are et al. 2004). The examples cited in Table 1 illustrate a used to prepare and apply mycorrhizal fungal inoculum; and striking symmetry between positive and negative outcomes the technical sophistication of these approaches varies of many mycorrhizal functions, which underscores the greatly. The simplest method is to apply soils that are known importance of knowing the ecological context in which to contain propagules of desirable mycorrhizal fungi to areas mycorrhizal fungi are introduced (Abbott & Robson 1991). that either lack these fungi or contain very low population One recent review concluded that often AM fungi do not densities. This method is often used during reclamation improve the growth of plants in production agricultural operations when Ôliving topsoilÕ is added back to mining systems as they are currently managed, particularly when soil wastes to help restore biotic interactions (e.g. Paschke et al. phosphorus is not in limiting supply (Ryan & Graham 2003). This whole-community soil inoculum is undefined, 2002). Similarly, meta-analysis has demonstrated an average and much more is added than the mycorrhizal fungi, positive effect on crop yield of mycorrhizal colonization, possibly including saprobic or pathogenic fungi, soil but suggested that such positive effects are much less likely invertebrates and prokaryotes. This approach may be when either soil P or indigenous mycorrhizal inoculum desirable in mine lands and other areas that lack functioning potential are high (Lekberg & Koide 2005). soil biota; however, other applications may require more There is evidence that in some systems, certain species of precise application of mycorrhizal fungi. mycorrhizal fungi may actually be detrimental to their hosts. Production of mycorrhizal inoculum for commercial For example, Glomus macrocarpum was shown to be the causal purposes has evolved considerably in recent years (Douds agent of stunting in tobacco (Modjo & Hendrix 1986; et al. 2000; Gianinazzi & Vosatka 2004) ranging from fungal Hendrix et al. 1992); and yield decline associated with propagation in on-site nursery plots (Sieverding 1991; Douds continuous cropping of corn and soybean has been linked to et al. in press) to axenic in vitro production in root organ particular AM fungi (Johnson et al. 1992). Similarly, inocu- culture (Adholeya et al. 2005) and liquid fermentation in lation with the EM fungi Laccaria proxima and Thelephora bioreactors (Rossi et al. 2002). In all of these preparations, the terrestris isolate TT3 resulted in growth depressions of Sitka source of the fungi is of critical concern, both in terms of the spruce 6 years after outplanting into natural soils with low beneficial performance of the symbiosis, and in the potential additions of phosphate (Le Tacon et al. 1992). The risks associated with the product use. If proper hygiene is not likelihood that inoculation with EM fungi will improve tree practiced during inoculum production, there is a high risk of performance following planting for reforestation appears to accidentally transferring pests or pathogens along with be highly dependent on ecological context (Bledsoe et al. mycorrhizal inocula (Douds et al. 2000). Gianinazzi & 1982; Perry et al. 1987; Castellano 1996). Vosatka (2004) stress the importance of instituting indus- This variance in mycorrhizal function is cause for concern try-wide quality control measures to ensure the production of because the purpose of including the fungi in commercially viable mycorrhizae that meet the expected requirements of produced mycorrhizal products is to capitalize on their end-users and are free from agents (e.g. pests) that might abilities to promote plant growth and survival across a negatively affect normal plant growth and development. narrow range of environments. However, only fungal isolates that are most conducive to large-scale production will be included in these products. There is no reason to POTENTIAL CONCERNS assume that production efficacy of a fungus corresponds We highlight three potential concerns associated with with its ability to increase host plant vigour. inoculation with mycorrhizal fungi: undesirable direct consequences for host plants in managed systems; direct Biodiversity concerns and indirect negative consequences to biodiversity; and negative consequences to ecosystem function. Introduced mycorrhizal fungi may directly impact local diversity of fungal communities and indirectly impact plant community composition. There are no documented cases of Undesirable direct consequences for crop production, introduced AM fungi facilitating the spread of invasive horticulture and forestry herbaceous plants, but given the widespread and general Inoculum that is intended to increase plant production and associations of these fungi with vascular plants, there are fitness may, in some cases, actually reduce it. Although there few locations where potential plant invaders are limited by are many reports of mycorrhizal enhancement of crop yields access to AM fungi. However, introduced AM fungi may and tree survival (e.g. Perry et al. 1987; Bethlenfalvay & contribute to plant invasions if invasive plants benefit more

Ó 2006 Blackwell Publishing Ltd/CNRS Global transport of mycorrhizal fungal inoculum 505 from introduced AM fungi than the native plant species, an Fruiting body observations and molecular analyses revealed important point to consider when applying AM fungal that EM fungi introduced with Eucalyptus in Spain were inoculum in restoration efforts. It appears that Bromus present on native shrubs (Diez 2005). Similarly, Amanita tectorum may more readily invade sagebrush steppe of the muscaria is now associated with Nothofagus forests in United States when forming arbuscular mycorrhizae, Tasmania and New Zealand, presumably as a consequence whereas individual plant growth is greater in isolation when of its introduction with pines (Fuhrer & Robinson 1992, lacking a mycorrhizae (Richardson et al. 2000). In addition, http://www.landcareresearch.co.nz/research/biosecurity/ competitive exclusion of native grasses by spotted knap- fungal/). weed may involve facilitation by AM fungi (Marler et al. Even if care is taken to introduce fungal species that 1999). may already be present in native habitats, problems may Facilitation of invasive plants by mycorrhizal fungi may still arise. Novel genotypes may outcompete native be more likely with EM fungi (Richardson et al. 2000). This genotypes and spread beyond the site of introduction, potential is perhaps best illustrated among the EM fungi and may interact differently than native genotypes with that were introduced along with their host plants for the native hosts, soil communities, and abiotic conditions. establishment of pine and eucalypt plantations. Nineteen Different strains of mycorrhizal fungi vary widely in their species of Pinus are considered problem invasives in the responses to the environment and in the benefits they southern hemisphere (Higgins & Richardson 1998), and provide to host plants (e.g. Cairney 2002), and there is members of the genus Eucalyptus are included on invasive evidence that some local genotypes of mycorrhizal fungi weed lists in the US and Europe (Warner 1999; Diez 2005). may be better adapted to their native environment and/or For example, Monterey pine (Pinus radiata) has a restricted may provide greater benefits to their native host plants native distribution in California and Mexico, but has been than non-local genotypes (e.g. Gildon & Tinker 1983; Stahl widely planted for agroforestry, especially in Spain, New & Smith 1984). If novel genotypes outcompete local Zealand, South America, and Australia where it now covers strains, locally adapted combinations of fungi and their more than 4 million hectares (Rogers 2002). Similarly, host plants may be disrupted. This disruption could also several species of Eucalyptus native to Australia have been occur through introgression between native and non-native introduced to North America, South America, Asia and fungal strains, if native and non-native strains are Europe (Richardson 1998). Successful introduction of these vegetatively or sexually compatible with each other. For trees required that EM fungi be imported, providing early example, North American L. bicolor strains used to evidence of the importance of the symbiosis to host trees inoculate Douglas fir plantation trees in Europe have (Smith & Read 1997). The application of EM inoculum can been shown to be genetically distinct from, but sexually be viewed as positive for agroforestry operations; however, compatible with, European strains at these sites (Mueller & there may also be unintended negative consequences if these Gardes 1991; De La Bastide et al. 1995). Hybridization or fungal introductions contribute to the spread of their introgression between introduced and native populations introduced host plants beyond plantation sites into neigh- of plants and animals has been shown to have significant bouring habitats (Richardson et al. 2000). For example, negative consequences for the native populations, inclu- eucalypts in Spain have become invasive in areas near large ding extinction (Rhymer & Simberloff 1996), especially forestry plantations (Diez 2005). These eucalypts are when the native populations are small or rare. colonized almost exclusively by fungal species or strains of Australian origin (Diez 2005). Ecosystem function The direct impacts of fungal introductions on native fungal communities are also important to consider. Several At the international scale, there is increasing interest in the studies have shown that exotic EM fungi are highly establishment of tree plantations to sequester carbon persistent in their novel environments (e.g. De La Bastide dioxide from the atmosphere. These forestation plans et al. 1995; Selosse 1997; Selosse et al. 1998a,b, 1999). For frequently include exotic trees (e.g. the BioCarbonFund, example, Laccaria bicolor isolates from North America were http://carbonfinance.org/biocarbon/router.cfm). The in- detected in Douglas fir (Pseudotsuga menziesii) plantations in troduction of a more diverse community of EM fungi has Europe 10 years after inoculation of out-planted seedlings been proposed to improve yield of trees in these plantations (Selosse et al. 1998a,b), and were also found to colonize (Dell et al. 2002). However, Chapela et al. (2001) have shown nearby uninoculated trees (Selosse et al. 1999). Isolates of that the EM fungus, Suillus luteus, introduced with Monterey Amanita muscaria have survived for > 36 years in Pinus pine into Ecuador grasslands, contributed to the removal of radiata plantations in Australia (Sawyer et al. 2001). In up to 30% of stored soil carbon in less than 20 years. Stable addition, exotic EM fungi may establish on native hosts and radioactive carbon isotope analyses revealed that Suillus where they could alter the distribution of native EM fungi. luteus utilized stored carbon to support abundant sporocarp

Ó 2006 Blackwell Publishing Ltd/CNRS 506 M. W. Schwartz et al. production, while plantation trees performed poorly (Cha- (James 1991; Bohlen et al. 2004). We know even less about pela et al. 2001). This dramatic impact on the soil carbon microbes in natural environments (e.g. Galan & Moreno cycle was not consistent with the biology of the fungus in its 1998). We often do not know, for example, exactly when or native habitat. Notably, S. luteus does not associate with how particular fungi have been introduced, or sometimes Monterey Pine in California (E.C. Vellinga, personal even if they are native or introduced in particular places communication). Further, sporocarp abundance in Ecuador (Orlovich & Cairney 2004; Pringle & Vellinga, 2006). was threefold greater than that of all sporocarp species There are at least four problems associated with combined in native California habitats (Chapela et al. 2001). diagnosing introductions of mycorrhizal fungi. Foremost is This simple example suggests the possibility of negative that identifying species of fungi can be difficult. Tradition- consequences of introduced mycorrhizal fungi on ecosystem ally mycologists have relied on morphological species functioning under some circumstances. concepts but abundant evidence demonstrates that morphological species possess cryptic reproductively isolated (Perkins & Raju 1986; Dettman et al. 2003) or genetic Assessment of risks associated with species introductions species (Koufopanou et al. 1997; Dettman et al. 2003; Ecologists have long tried to ascertain predictable ecological Pringle et al. 2005; Taylor et al., in press). When species patterns in the propensity of introduced species to become are defined according to morphology, often for practical costly noxious invaders. Attempts to identify universal traits purposes, what are identified as different ÔecotypesÕ of the of successful invaders (e.g. Baker 1965) have generally failed same morphospecies can function very differently (e.g. Stahl but attempts to understand traits that predict the invasive & Smith 1984; Bethlenfalvay et al. 1989). Invasive species potential of smaller, more constrained suites of species have may be difficult to identify because the concept of a fungal met with more success (e.g. Rejmanek & Richardson 1996; species can vary among biologists. AM fungi pose a unique Reichard & Hamilton 1997; Kolar & Lodge 2001). challenge because they are often defined according to Thinking very generally about the potential of non-native morphology, but the genetic system is a focus of ongoing biota to cause ecological harm, we know that: (1) research (Pawlowska & Taylor 2004; Hijri & Sanders 2005). numerically, most introductions fail (Simberloff & Stiling The individual nuclei within a single morphologically 1996; Mack et al. 2000); (2) among the species that defined species of AM fungus may differ, for example, successfully establish, most are relatively innocuous and making it possible for an introduced nuclear type to do not require costly management responses (Hiebert 1997); introgress into a native population of nuclei, even if the (3) for species that establish and create costly problems morphotype does not establish or invade. This kind of there is often a lag time between introduction and ecological invasion is rarely considered by ecologists but is exactly damage (Mack et al. 2000; Sakai et al. 2001); (4) invasive analogous to the introgression of genes after hybridization species and their novel interactions with the existing biota events, or the horizontal transmission of genes between can result in strong selection, rapid evolution and novel and bacterial lineages. In other kingdoms hybridization may unpredictable interspecific interactions (Parker & Gilbert serve as a stimulus to invasion (Ellstrand & Schierenbeck 2004); and (5) noxious problem species cost societies 2000; Ayres et al. 2004; Petit 2004). billions of dollars per year (U.S. Congress Office of Second, because soil is a cryptic environment, it has Technology Assessment 1993; Naylor 2000; Pimentel et al. proven difficult to assess the abundance and distribution of 2000). As a result, there is great value in identifying where mycorrhizal fungi (Johnson et al. 1999). We lack adequate the low probability but exceedingly costly problem intro- knowledge of the biogeography of mycorrhizal fungi ductions may occur and working to adopt management (Pringle and Vellinga, 2006). Available studies suggest that practices that minimize the likelihood of these situations many AM fungi, and some EM fungi, are remarkably (Mack et al. 2000; Mack 2000). cosmopolitan in their distributions (Molina et al. 1992; Generally speaking, the larger the species, the more likely Morton & Bentivenga 1994; Stutz et al. 2000). Once again, we know the timing, source and consequences of species however, there is a conflict between morphological and introductions. We may know, for example, when, where and genetic species concepts. When species are defined accord- why various vertebrates were introduced, areas where they ing to reproductive or genetic isolation the different cryptic are now invasive, as well as their rate of spread (Shigesada & lineages generally have constrained distributions (Petersen & Kawasaki 1997; Abbott 2002). The corollary to this Hughes 1999; Taylor et al., 2000). Only one fungus has been observation is that the smaller the organism, the less, in demonstrated to possess cryptic genetic species with global general, we know about invasions. Relatively little is known, distributions (Pringle et al. 2005). Global distributions of for example, regarding the invasion of earthworms to North reproductively isolated or genetically defined species are an America, despite our current understanding of the dramatic exception. Additional biogeographical data are crucial to our ecosystem effects that they have once they are established understanding of invasive fungi. If morphological species

Ó 2006 Blackwell Publishing Ltd/CNRS Global transport of mycorrhizal fungal inoculum 507 are globally distributed, the intentional movement of ability, and competitiveness (Grime 2001). Some of these mycorrhizal fungi may be of little concern. Alternatively, if life-history traits can be loosely associated with the potential cryptic species are endemic, then they are susceptible to for noxious behaviour. For example, a large number of displacement and extinction by introduced types. problem invasive plant species are disturbance dependent, Third, information on the endemic diversity of especially ruderal or weedy species with high dispersal but low AM fungal communities is sparse. For example, one competitive capacity (Kolar & Lodge 2001; Sakai et al. intensively sampled North Carolina field possessed at least 2001). Among the worst of these invaders, however, are 37 AM fungal species; one-third of these species were species (e.g. spotted knapweed, star thistle, purple loose- discovered at that site and have not been found elsewhere strife) that carry attributes that allow them to rapidly invade (Bever et al. 2001). It will be impossible to understand how disturbed habitats and maintain competitive dominance fungal communities change without an understanding of through time. Some of the most costly and difficult to baseline community composition. contain invasive species are the relatively few that success- Fourth, perhaps because of the difficulties associated with fully invade mature vegetation in relatively undisturbed defining species, assessing fungal biogeography, and descri- habitats (e.g. garlic mustard and leafy spurge; Meekins & bing fungal communities, there has been little effort to track McCarthy 2001). inadvertent international transport of mycorrhizal fungi Aspects of life history may be used to predict relative found in horticultural products or through other means of risks associated with introductions to novel ecosystems. For transport (Perrings et al. 2002). These challenges result in a example, many EM species produce small airborne spores great deal of difficulty discerning whether or not mycorrh- that are likely to be circulated widely, while some EM and izal fungi are native to a particular location. Mycologists nearly all AM fungi produce belowground spores which are need to further develop and apply molecular techniques that distributed only locally by animals and physical soil will allow us to track the establishment, invasion and movement (Allen 1991). The risk of spread via spore persistence of fungal isolates in novel environments (for an dispersal into nearby non-target habitats is expected to be example with EM fungi, see Selosse et al. 1998a,b, 1999). significantly different among these groups of fungi. In Gianinazzi & Vosatka (2004) conclude that current DNA addition, recent theoretical studies of plant–parasite inter- technologies for tracking inoculated AM fungi only allow actions suggest that parasites with higher rates of gene flow detection at the species level (where species is defined may be better able to adapt to local host populations, as long according to morphology), and stress the need for further as gene flow does not completely homogenize parasite development of ÔstrainÕ-specific probes and the construction populations (Gandon & Michalakis 2002; Morgan et al. of kits to better track the persistence of commercially 2005). If these results are applicable to the mycorrhizal produced mycorrhizal inoculum. Towards this goal, Ôbar- symbiosis, then differing dispersal abilities among types of codingÕ technology may be important for future research mycorrhizal fungi may help predict the potential for non- (http://www.barcoding.si.edu). native populations to adapt to novel environments. Evidence in support of the importance of life-history attributes to predict invasion success comes from studies of PREDICTING TRAITS OF INVASIVE AND NON- EM fungi, where invasion may be less common for species INVASIVE MYCORRHIZAL FUNGI with hypogeous or closed sporocarps and more likely for Mycorrhizal fungi vary widely with respect to life-history species with open sporocarps and wind or insect dispersed attributes and ecological aspects of their interactions with spores. Truffles and truffle-like fungi typically possess plant hosts (Table 2). Ultimately, we may be able to use closed sporocarps, and such fungi have been introduced our knowledge of life-history attributes such as host across continents at multiple times (Dennis 1975; Sogg specificity, competitive ability and dispersal mode to infer 2000; Trappe & Ca´zares 2000; Fogel & States 2001; Yun & the likely ability of mycorrhizal fungi to become an Hall 2004). Nevertheless, the available (albeit limited) data invasive problem. This knowledge may allow us to develop suggest that these taxa are not invasive. general predictors of how and when undesirable AM and In contrast, gilled and poroid mushrooms possess EM fungi may establish and spread to non-target species forcibly discharged spores that may be carried long distances and habitats. by wind or in some cases, flying insects. Two obvious examples of invasive mycorrhizal fungi are Amanita muscaria (Bougher 1996; Orlovich & Cairney 2004) and Amanita Life-history traits phalloides (A. Pringle, unpublished data); both species make Ecologists have identified a continuum of life-history traits gilled mushrooms with airborne spores. An estimate of how in reproduction (allocation to numerous, small propagules quickly species with open sporocarps will travel can be made vs. few, larger and well-provisioned propagules), dispersal using data of the saprobe Clathrus archeri (Parent et al. 2000).

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This fungus, whose spores are carried by insects, appeared clearly more competitive than other species. For example, in the Alsace region of France in 1920 and by 1999 (and Hepper et al. (1988) showed that Glomus caledonium was more perhaps earlier) C. archeri had travelled to and established in competitive than Glomus mosseae, and both species were the Galicia region of Spain, a distance of at least 1400 km in much more competitive than the Glomus isolate known as c. 70 years. ÔE3Õ. Presence of one fungus in a root system can alter the ability of another to colonize roots (Pearson et al. 1993) but this can depend on the stage in the life cycle of the fungi Colonization and competition (Pearson & Schweiger 1993). Colonization of roots by AM Some ectomycorrhizal fungi have been shown to have fungi is a complex phenomenon and seasonal dynamics different abilities to compete for space on the roots of their (Merryweather & Fitter 1998) demonstrate that simple host plants. Kennedy & Bruns (2005) showed that assessments at one point in time would offer incomplete Rhizopogon occidentalis, a ruderal EM fungus typically found descriptions of colonization success or failure. only on the roots of pine seedlings in relatively early stages Outcomes of competition among mycorrhizal fungi at of plant succession (Horton et al. 1998; Baar et al. 1999), decadal and longer time scales will ultimately be most more quickly colonized host roots than Rhizopogon salebrosus, relevant for determining whether introduced mycorrhizal which exhibits some ruderal behaviour but is also found on fungi can establish and persist in non-target ecosystems. We the roots of trees in mature forest. Interestingly, rapid need to understand whether there are consistent tradeoffs colonization by R. occidentalis seemed to give it a priority among species for initial colonization ability, long-term effect in competitive interactions with R. salebrosus, with competitive ability, dispersal ability, and benefits to host R. occidentalis inhibiting colonization by R. salebrosus in mixed plants. For example, more local dispersal, as in fungi with species treatments. This example illustrates an important closed sporocarps, may be associated with higher levels of distinction between different scales of colonization ability ecotypic variation and host specialization. Unfortunately, for mycorrhizal fungi – Rhizopogon species produce below- very few data exist to assess such tradeoffs. It is conceivable ground sporocarps that are dispersed locally, and thus would that the ability to compete for root space may favour long- be considered to have more limited colonization ability than term persistence by a fungus, but it may not be a good aerially dispersed species. However, within local popula- predictor of the ability to spread in an ecosystem, or of its tions, species such as R. occidentalis exhibit ruderal charac- influence on host plant fitness. For example, it may be that teristics, quickly colonizing host plants through rapid spore the mycorrhizal fungal species that have the best initial germination after disturbances. Rhizopogon species have been colonization ability, and thus are desirable for ease of used to inoculate ectomycorrhizal plants for commercial inoculation, also tend to have poor long-term competitive purposes, and a species such as R. occidentalis seems to ability, a relatively low growth benefit to host plants, and/or possess a number of desirable characteristics for such uses: a high ability to spread into non-target plant communities, in rapid colonization, initial competitive ability due to priority which case these fungi would be undesirable from a effects, and inability to persist in later stages of succession; management perspective. however, for any particular management scenario, it would also be crucial to know whether inoculation with R. CONCLUSIONS AND RECOMMENDATIONS occidentalis is beneficial to the target host plant. Similarly, AM fungi differ in the rates at which they We believe that evidence suggests that there is clear colonize plant roots as well as their abilities to compete with potential for non-indigenous mycorrhizal fungi to persist other AM fungi once they are inside roots. For example, and invade non-target habitats. These invasions may have Hart & Reader (2002) showed that isolates from the positive, neutral, or negative effects on plant growth, local Glomaceae tend to colonize roots significantly more quickly, fungal and plant communities and ecosystem processes. usually within 3 weeks, compared with isolates from the Figure 1 summarizes these potential effects, and provides a Acaulosporaceae and Gigasporaceae, some of which took general framework of testable hypotheses. We can no longer up to 8 weeks. Studies of interactions among indigenous assume that all interactions with mycorrhizal fungi will result and introduced species of AM fungi show that competitive in positive or negligible effects. With ecological studies outcomes also vary with fungal taxa (Hepper et al. 1988), as documenting the potential for serious negative by-product well as proximity of the fungal propagules relative to plant consequences of inoculation, more attention needs to be roots (Lopez-Aguillon & Mosse 1987), population densities placed on research that can help elucidate best management (Abbott & Robson 1981), and the presence or absence of practices for mycorrhizal treatments. Those applying fungal hyperparasites in the system (Ross & Ruttencutter 1977). treatments should expect a range of outcomes from positive Although competitive outcomes may sometimes depend on to negative within natural systems as well as in managed initial relative inoculum densities, some taxa of AM fungi are systems. Thus, careful consideration of need and techniques

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Figure 1 A conceptual model illustrating issues of primary concern when using fungal inoculum in plant management. The top row represents traits of non-native mycorrhizal inoculum that are hypothesized to be associated with impacts on native communities and ecosystem processes. These are often the very same traits sought after as favourable attributes for management use. The middle row represents three primary areas of potential impacts and their interactions. The bottom row represents mechanisms by which fungal inocula may either directly or indirectly affect natural systems. are warranted. We make three specific recommendations mycorrhizal inoculum containing indigenous fungi (Abbott with this in mind. et al. 1992; Douds et al. 2000). On-farm production of AM First, the primary consideration for each proposed fungal inoculum is feasible in most situations and it is highly application of mycorrhizal inoculation should be whether desirable because it minimizes production costs (Douds or not inoculation is necessary (Abbott & Robson 1991). et al. 2000). Also, producing inoculum locally will help Mycorrhizal fungi are ubiquitous and abundant in most minimize the potential risk of spreading non-indigenous agricultural systems (Olsson et al. 1999); consequently, pathogens and pests that may accidentally contaminate inoculation is generally not necessary to produce mycorrh- commercial mycorrhizal inoculum products. During every izae on crop roots. In addition, ecotypes of AM fungi in step of the process, care should be taken to ensure the systems with a history of high fertilizer inputs may not be production of pest-free mycorrhizal inoculum (Menge beneficial to plant growth (Johnson 1993, Bell et al. 2003). 1984). Alternatively, population densities of indigenous AM fungi Our third recommendation pertains to the use of nonin soils managed for agriculture and horticulture may be indigenous fungi. Local strains or species may be unavailable severely depressed due to soil sterilization, tillage and fallow or may be known to be incompatible with the target plant (Douds & Johnson 2003). Both AM and EM fungi may be species in many managed systems. Mixed strain AM inocula eliminated from severely disturbed ecosystems such as mine might be viewed as increasing the probability of a positive lands or eroded slopes so that inoculation with mycorrhizal target effect, but this strategy also carries risk. Strains fungi is necessary for successful reclamation or restoration beneficial to target plant growth do not always dominate and (Jasper et al. 1987; Lumini et al. 1994). Finally, reforestation mixed inocula increase the likelihood of an unintended projects often benefit from the addition of EM inoculum negative consequences, such as non-target invasion. As when the abundance and diversity of natural inoculum is discussed above, when EM plants are planted as exotics for low due to previous land uses (Le Tacon et al. 1992). timber production, compatible exotic EM fungi are often Second, we recommend adopting policies that favour the introduced with them to ensure successful establishment. If use of local mycorrhizal types, where feasible. A conserva- non-indigenous fungi must be used, then steps should be tive approach to managing biotic integrity is to recommend taken to minimize the risk of introducing mycorrhizal fungi managing indigenous mycorrhizal fungi that are already that could become problem invasive species. For such present in the soil (Trappe 1977; Abbott & Robson 1982; situations, we propose that the isolates used for inoculation Sylvia & Burks 1988; Bethlenfalvay & Linderman 1992; should be selected to have the following traits when Jasper 1994; Parlade et al. 1996; Berman & Bledsoe 1998; possible: Douds et al. 2000). When mycorrhizal fungal propagules are (1) High benefit to the target host plant. absent, or in extremely low densities, then inoculum (2) High specificity to the target host plant species. containing local strains of mycorrhizal fungi should be (3) Among EM fungi, low ability to utilize non-host developed and utilized to the extent possible. Protocols are carbon sources, in order to minimize the opportunity already in place for the selection and production of

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Table 3 Research needs in mycorrhizal fungi needed to better predict invasion potential. There is currently insufﬁcient knowledge to accurately predict ecological outcomes of inoculation with non-native isolates of mycorrhizal fungi. Future research is needed to develop assessment methods and provide ecological information to ensure inoculation safety

Assessment methods Develop protocols for rapid determination of the taxonomy of mycorrhizal fungi, with complementary use of morphological and molecular methods Develop protocols for determining the biogeographical distribution of mycorrhizal fungi, especially cryptic species Develop protocols for tracking speciﬁc genotypes of mycorrhizal fungi in novel environments Develop protocols for assessing the infectivity of inoculum of mycorrhizal fungi Develop protocols for screening of inoculum for the presence of pests and pathogens Ecological information Assess life history traits of fungal taxa that are used as inoculum, particularly: host range, competitive ability against other mycorrhizal fungi and other fungal taxa, dispersal ability, and average impact on the growth of multiple host plant species, and comparative infectivity of fungi from different quantities of inocula Characterize local communities of mycorrhizal fungi, especially in habitats near areas of frequent mycorrhizal inoculation Determine the degree of compatibility of mycorrhizal fungi with invasive plant species in non-native environments Assess potential impacts of colonization by mycorrhizal fungi on soil carbon storage

for EM fungi to exist as partial saprobes in introduced plant species and ecosystem processes. It is of primary habitats. importance that the potential benefits of mycorrhizal inocu- (4) Rapid colonization ability, for ease of inoculation. lation be balanced with the potential costs of unwanted (5) Low dispersal ability, to reduce the potential for invasions. Policies that reduce the likelihood of ecologically encroachment into non-target habitats. costly introductions are unlikely unless mycorrhizal ecologists (6) Poor long-term competitive ability which would allow can develop a much better understanding of the ecological inoculation and establishment of host plants, followed costs incurred by continuing current practices, and develop by extirpation of the introduced fungus by native fungi. feasible alternative strategies to inoculation with non-local strains. However, until we develop better empirically based Note that recommendations (2), (3), (5) and (6) would not support for these concerns, as well as models for manage- apply if the management treatment utilizes indigenous fungi ment, there is likely to be little impetus to alter management as inoculum because traits such as high dispersal ability and strategies. With inoculation treatments increasing, and new long-term competitive ability would then be desirable for companies emerging to meet the demand for these products, building a sustainable ecosystem. mycorrhizal ecologists must respond quickly to fill the Unfortunately, we do not have sufficient data on all six of research void that currently exists in order to assess the need these traits for specific fungal strains to allow selection of for the cautious approach that we advocate here. These fungi based on these traits. More research is necessary to research gaps can be closed with comparative studies linking increase our knowledge of the ecological attributes of phylogenetic relatedness, life-history traits, and ecological mycorrhizal fungi, and our ability to assess persistence and effects in commonly used fungal types, careful field studies of spread by non-indigenous mycorrhizal fungi (Table 3). potential spread of fungi to non-target hosts, and a synthesis Research in these areas will allow us to better predict of the range of target effects expected from inoculation. This inoculation and growth responses as well as unwanted is an achievable research agenda. Several research groups are invasions into native ecosystems (Hart et al. 2001). We need currently working on aspects of these problems. Care is to know whether the fungi that are being used most frequently needed to make sure that this emerging science is integrated in inoculation efforts tend to have positive benefits for the into mycorrhizal management. target host plant and high host specificity, the ability to compete with native fungi in the short and long term, and the ability to spread into and affect non-target plant species or ACKNOWLEDGEMENTS habitats. Ultimately, scientists working on invasion biology This work was conducted as a part of the Narrowing the Gap and ecosystem health will need to work towards thoroughly Between Theory and Practice in Mycorrhizal Management documenting cases of non-native fungal impacts on native

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Working Group supported by the National Center for Bell, J., Wells, S., Jasper, D.A. & Abbott, L.K. (2003). Field in- Ecological Analysis and Synthesis, a center supported by the oculation with arbuscular mycorrhizal fungi in rehabilitation of National Science Foundation (Grant #DEB-00-72909); the mine sites with native vegetation, including Acacia spp. Aust. Syst. Bot. University of California at Santa Barbara; and the State of , 16, 131–138. Berman, J.T. & Bledsoe, C.S. (1998). Soil transfers from valley oak California. This work was also supported by NSF grants (Quercus lobata Nee) stands increase ectomycorrhizal diversity and DEB0415563 to C. Gehring and DEB0316136 to N. alter root and shoot growth on valley oak seedlings. Mycorrhiza, Johnson, a BLM grant to N. Johnson (JSA990018), and by 7, 223–235. a Discovery grant from the Natural Sciences and Engineering Bethlenfalvay, G.J. & Linderman, R.G. (eds) (1992). Mycorrhizae in Research Council of Canada to J. Klironomos. We thank G. 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Ó 2006 Blackwell Publishing Ltd/CNRS Managing arbuscular mycorrhizal fungi in cropping systems

C. Plenchette1, C. Clermont-Dauphin2, J. M. Meynard3, and J. A. Fortin4

1INRA, UMR BGA, Dijon, France (e-mail: [email protected]); 2INRA, UMR APC, Guadeloupe, France; 3INRA, SAD, Thiverval-Grignon, France; and 4Biologiste Conseil Inc., Loretteville, Québec, Canada. Received 16 September 2003, accepted 26 May 2004.

Plencette, C., Clermont-Dauphin, C., Meynard, J. M. and Fortin, J. A. 2005. Managing arbuscular mycorrhizal fungi in cropping systems. Can. J. Plant Sci. 85: 31–40. Market globalization, demographic pressure, and environmental degradation have led us to reconsider many of our current agricultural systems. The heavy use of chemical inputs, including fertilizers and pesticides, has resulted in pollution, decreased biodiversity in intensively-farmed regions, degradation of fragile agro-ecosystems, and prohibitive costs for many farmers. Low input sustainable cropping systems should replace conventional agriculture, but this requires a more comprehensive understanding of the biological interactions within agro-ecosystems. Mycorrhizal fungi appear to be the most important telluric organisms to consider. Mycorrhizae, which result from a symbiosis between these fungi and plant roots, are directly involved in plant mineral nutrition, the control of plant pathogens, and drought tolerance. Most horticultural and crop plants are symbiotic with arbuscular mycorrhizal fungi. Mycorrhizal literature is abundant, showing that stimulation of plant growth can be mainly attributed to improved phosphorous nutrition. Although the mycorrhizal potential of its symbiosis to improve crop production is widely recognized, it is not implemented in agricultural systems. There is an urgent need to improve and widely apply analytical methods to evaluate characteristics such as, relative field mycorrhizal dependency, soil mycorrhizal infectivity, and mycorrhizal receptivity of soil. Decreased use of fertilizers, pesticides, and tillage will favour arbuscular mycorrhizal fungi. However, shifting from one system to a more sustainable one is not easy since all components of the cropping system are closely linked. Different cases, from actual agricultural practices in different countries, are analyzed to highlight situations in which mycorrhizae might or might not play a role in developing more sustainable agriculture.

Key words: Cropping systems, mycorrhizae, sustainability, technical itineraries, rotation

Plencette, C., Clermont-Dauphin, C., Meynard, J. M. et Fortin, J. A. 2005. Gestion des champignons mycorchiziens a arbuscules dans les systemes de culture. Can. J. Plant Sci. 85: 31–40. La globalisation mondiale du marché, la pression démographique et la dégradation de l’environnement ont, dans plusieurs parties du monde, mené à une réévaluation des systèmes agricoles actuels. Les modifications de l’environnement montrent que l’utilisation des engrais et des pesticides a atteint ses limites : pollution et perte

For personal use only. de biodiversité dans les régions d’agriculture intensive, dégradation des systèmes agricoles les plus fragiles, coûts prohibitifs pour les producteurs les plus pauvres. L’agriculture conventionnelle doit s’orienter vers des pratiques plus durables, mais les systèmes de culture durable à faibles intrants ne pourront être viables que par une meilleure connaissance et maîtrise des interactions biologiques dans ces agro-systèmes. Les champignons mycorhiziens apparaissent comme les organismes telluriques les plus importants à prendre en considération. Les mycorhizes qui résultent de l’association de ces champignons symbiotiques avec les racines des plantes sont directement impliquées dans la nutrition minérale, l’absorption de l’eau et la protection contre certains agents pathogènes. La plupart des plantes horticoles et de grande culture forment des mycorhizes. La littérature sur les mycorhizes est de plus en plus abondante, leur rôle dans la stimulation de croissance des plantes étant attribué principalement à une meilleure nutrition phosphatée. Cependant, si le potentiel des mycorhizes est reconnu, leur prise en compte dans les systèmes de culture reste à faire. Il est donc nécessaire d’améliorer et d’appliquer les méthodes analytiques permettant d’évaluer les paramètres fondamen- taux tels que la Dépendance Mycorhizienne Relative au Champ des plantes, le Potentiel Infectieux Mycorhizogène du sol, la Réceptivité du sol aux champignons mycorhiziens. Moins d’engrais, de pesticides et de travail du sol sont favorables au développe- ment des mycorhizes et donc au maintien du potentiel infectieux mycorhizogène du sol. Mais le passage des systèmes actuels vers des systèmes plus durables n’est pas simple car les composantes des systèmes de culture sont étroitement liées les unes aux autres. Quelques systèmes actuellement pratiqués dans différents pays sont analysés pour illustrer les situations dans lesquelles les Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 122.56.27.149 on 11/27/18 mycorhizes pourront ou non jouer un rôle pour le développement d’une agriculture plus durable.

Mots clés: Systèmes de culture, mycorhize, durabilité, itinéraires techniques, rotation

Agricultural productivity has increased steadily since the developing countries; the importance of agricultural prod- middle of the last century in temperate regions and, more ucts for trade in many countries; the competition between recently, in tropical areas (Craswell and Karjalainen 1990) urbanization and agriculture for land, and; the competition mainly due to improved varieties, machinery, fertilizers, and that is likely to develop between food and non-food produc- pesticides. Many factors have contributed to making this tion. However, the tools that are presently available for pro- increase in crop production essential, including: population ducers to increase yields tend to increase input costs, the increases worldwide (Alexandratos 2003); food shortages in number of operations in the field, and the financial risks of 31 32 CANADIAN JOURNAL OF PLANT SCIENCE

failure. Moreover, environmental degradation linked to the (1959), Daft and Nicolson (1966), Gerdemann (1968), and use of chemical inputs (i.e., water pollution from nitrates, Hayman and Mosse (1971), the scientific community has phosphates, and pesticides) is increasingly widespread and advocated greater use of AM fungi in agricultural practices sometimes irreversible. In some cases, secondary effects on (Gianinazzi and Schüepp 1994; Harrier and Watson 2003), biocenoses and soil impoverishment have weakened crop- particularly to reduce the use of P fertilizers, but so far this ping systems to make them increasingly dependent on has not happened. The intensive agriculture that developed chemicals (Clermont-Dauphin and Meynard 1997). from the mid-20th century was based on new cultivars and Growing societal demands for clean agriculture, high-quali- an increased use of fertilizers and biocides. As fertilizer and ty food, and more information on how food is produced are pesticide applications decrease the development of AM finally having an effect on decreasing the level of chemical fungi and mycorrhizae, their effects would have been inputs used in developed countries (Boiffin et al. 2001). In severely limited. In order to be beneficial, AM fungi must developed countries, therefore, we can safely predict that first be present in the soil (indigenous) or introduced (select- agriculture will become less intensive. In developing coun- ed strains), and secondly, they must be protected and/or tries, however, the reverse is true. In these countries the improved by cultural practices. Presently, most cropping great need for food implies a trend toward intensification, systems used worldwide do not protect AM fungi and favor mainly through the use of more fertilizers (Norse 2003). mycorrhizal development. A more sustainable agriculture that is “ecologically Cropping systems vary greatly throughout the world, sound, economically viable, socially just and humane” from simple monoculture to complex intercropping systems. (Gips 1987) should aim to recycle minerals in the soil with This paper examines the effects of select cropping systems no or few external inputs, maintain a high biodiversity in the on AM fungi. Based on an analysis of some temperate and agro-ecosystems, favour mechanical and biological weed tropical cropping systems, and on a review of agronomic control, and better exploit soil-plant-microbe interactions practices that are detrimental or beneficial to AM fungi, we for plant nutrition and protection against pests (Edwards et shall discuss recent changes in cropping systems and new al. 1990). opportunities to increase the efficiency of AM symbiosis. Beneficial micro-organisms, including soil-borne symbionts, N2-fixing bacteria, and arbuscular mycorrhizal (AM) ARBUSCULAR MYCORRHIZAL FUNGI IN fungi, provide minerals to plants and are directly implicated CROPPING SYSTEMS in crop production. Mycorrhizae provide mutualistic symbiosis between some soil-borne fungi and plant roots (Frank Fundamental Concepts 1885). Symbiotic fungi are common worldwide in all soil In order to examine how AM fungi can benefit field crops types and climates; AM fungi (Morton and Benny 1990), for and what conditions are required for their development, it is example, have coexisted and coevolved with plants for essential to understand that plants inoculated with AM fungi about 400 million years (Malloch et al. 1980; Pirozynski and differ markedly in their growth response (Daft and Nicolson 1969). This growth-stimulating effect, referred to as ‘myc- For personal use only. Dalpé 1989). This means most of the root systems of agricultural/horticultural plants and crops are colonized by AM orrhizal dependency’ (MD) (Gerdemann 1975), is highly fungi (Harley and Harley 1987; Sieverding 1991). variable (Sanders et al. 1977) and is influenced by several There is an abundance of literature on AM fungi, particu- factors such as plant species (Gerdemann 1968), soil fertili- larly on the beneficial effects of symbiosis on plant growth, ty (Hayman and Mosse 1971), and fungal strain (Plenchette which is mainly attributed to improved mineral nutrition et al. 1982). From an agronomic point of view, this makes it (Harley and Smith 1983). Arbuscular mycorrhizal fungi col- necessary to examine the mycorrhizal dependency of plants onize both plant and soil. In rhizospheric soil, these fungi under field conditions. This concept of relative field mycor- develop a hyphal network that serves as a fundamental link rhizal dependency, proposed by Plenchette et al. (1983), between the soil, the nutrient reservoir, and the plant. This takes into account many factors other than plant responsive- hyphal network is more efficient for ion uptake than root ness (growth difference between mycorrhizal and non-myc- hairs (Dorneless et al. 2001; Sanders et al. 1977). orrhizal plants). Phosphorus is the most common nutrient that is translocat- Since mycorrhizal dependency is a genotypic property of

Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 122.56.27.149 on 11/27/18 ed from the soil to plants through hyphae; hyphae can bridge plants (Janos 1993), it varies according to growing condi- the depletion zone that occurs around plant roots for immo- tions, although the relative response of each plant species is bile elements, such as phosphorus (Hatting et al. 1973). This maintained. For example, legumes are generally more mechanism is particularly important for the many soil types mycotrophic than grasses, and corn response to mycorrhizae that have low levels of bioavailable phosphorus (Hayman is always greater than that of wheat. This property is impor- and Mosse 1971. Arbuscular mycorrhizae can also provide tant because cropping systems usually include plants that many non-nutritional benefits to crops, including: allevia- have a range of mycorrhizal dependencies and even some tion of water (Safir et al. 1971; Allen and Boosalis 1983) plants that are not dependent at all, such as Chenopodiaceae and salt (Ruizlozano et al. 1996) stress, protection against and Brassicaceae, which do not form typical arbucular myc- some soil-borne pathogens (Baltruschat and Schönbeck orrhizae. Cropping systems that include highly mycorrhizal- 1972; Roncadori and Hussey 1977) and improvements to dependent plants, for example, can increase mycorrhizae in soil structure (Hamel et al. 1997; Schreiner and the soil and consequently increase AM fungi populations. Bethlenfalvay 1995). Since the pioneering work of Baylis To form mycorrhizae, plant roots need to come into contact PLENCHETTE ET AL. — MANAGING ARBUSCULAR MYCORRHIZAL FUNGI IN CROPPING SYSTEMS 33

with AM fungal propagules, which are soil-borne spores, centration that is twice as large as that of the wild species. soil hyphae, or root pieces bearing fungal structures (inter- For Johson and Pfleger (1992), there is no doubt that “crop nal hyphae or vesicles). Together, these propagules repre- breeding programs selecting for high yielding varieties sent the quantity of AM inoculum, i.e., the population of under fertilized conditions may inadvertently select geno- AM fungi in the soil. The concept of mycorrhizal soil infec- types that are unresponsive to mycorrhizae.” Along breed- tivity (Plenchette 1989) takes into account the quantity of ing programs it appears that plants lose some genes related AM propagules and the ability of the soil to favour their ger- to mutualism. Some modern lines of wheat, for example, mination. Mycorrhizal dependency of plants and mycor- have been shown to have a lower mycorrhizal dependency rhizal soil infectivity are reciprocally linked since the latter than their ancestors (Hetrick et al. 1992). It also appears that is a condition of the expression of the former which govern breeding maize for resistance to fungal pathogens produces the level of the latter. Thus, mycorrhizal development in the lines that are less mycotrophic than susceptible ones (Toth field is largely dependent on cropping systems, and in par- et al. 1990). Breeding for better symbiosis (Rengel 2002) ticular, the cropping sequence of plants that exhibit a range could be an objective for a sustainable agriculture that of mycorrhizal dependencies. would contribute to improved nutrient and water efficiency It was recently documented under field conditions that (Meynard and Jeuffroy 2002). fallow and cultivation of sunflower, maize, soybean, potato, There are numerous reports, sometimes conflicting, on wheat, sugar beet, and rape had an effect on AM coloniza- the effects of pesticides, particularly fungicides, on mycor- tion and the growth of a subsequent maize crop (Arihara and rhiza development. Generally, the effects are deleterious to Karasawa 2000; Karasawa et al. 2002). The lowest mycor- AM fungi, but vary depending on the active ingredient and rhizal development, tissue P content, and yield were found the rate of application (Schreiner and Bethlenfalvay 1997; in maize following rape, fallow, and sugar beet, then, in Nemec 1980). It has also been documented that some fungi- increasing order of maize yield, previous crops of wheat, cides (fosetyl-Al, metalaxyl, promamocarb) not only pre- potato, soybean, maize, and sunflower. The yield of maize serve but also stimulate AM fungi (Jalali and Domsch 1975; increased according to the mycorrhizal dependency of the Jabaji-Hare and Kendrick 1985, 1987; Rutto et al. 1999). preceding crops. Remarkably, the hierarchy of wheat, pota- Fungicides applied as seed coating (Spokes et al. 1989) are to, and maize is the same as previously found in another probably more detrimental to AM development than fungi- field experiment (Plenchette et al. 1983). This result agrees cides that are applied when plants are already mycorrhizal with the concept of mycorrhizal dependency of plants that is (Plenchette and Perrin 1992). fundamental to understanding the role of mycorrhizae in cropping systems. (ii) Soil Modifications Soil tillage affects the distribution of AM propagules in the Effects of Agricultural Practices soil (Smith 1978), and reduces mycorrhizal root coloniza- Several other factors that directly affect mycorrhizal devel- tion (Yocom et al. 1985) and early plant P uptake

For personal use only. opment arise from agricultural practices. These include: (i) (Vivekanandan and Fixen 1991). These detrimental effects plant modifications, such as breeding, pesticides, growth have been mainly attributed to the disruption of the hyphal regulators, and seed coating with fungicides, and; (ii) soil network remaining from the previous crop (O’Halloran et al. modifications, such as fertilization, pesticides, tillage, and 1986; Evans and Miller 1990). With a high inoculum densi- fallow. These effects are well documented elsewhere; the ty, however, soil disturbance has no effect on the AM colo- following sections provide a brief summary. nization of plants (McGonicle and Miller 2000). Arbuscular mycorrhizal colonization of plants (Hayman (i) Plant Modifications et al. 1975) and mycorrhizal soil infectivity are decreased by Breeding to obtain new varieties is a key component in both mineral and organic P fertilizers (Plenchette 1989). developing agricultural systems. Mycorrhizal dependency Plant mycorrhizal colonization and mycorrhizal dependency not only varies among crops (Plenchette et al. 1983; Khasa are negatively correlated with phosphorus concentration in et al. 1992; Duponnois et al. 2001a), but also among plant the soil solution (Habte and Manjunath 1987). The forma- species. This has been demonstrated for cultivars of wheat tion and beneficial effects of mycorrhizae can be obtained in

Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 122.56.27.149 on 11/27/18 (Azcon and Ocampo 1981), soybean (Heckman and Angle high P soils if these soils have a high P-ability (Plenchette 1987), corn (Toth et al. 1984), millet (Krishna et al. 1985), and Fardeau 1988). groundnut (Kesava Rao et al. 1990), banana (Declerck et al. Fumigants, such as methyl bromide, destroy all soil 1995a), and eucalyptus (Adjoud et al. 1996). organisms that are more sensitive than soil-borne pathogens, Breeding programs are generally conducted on experi- including AM fungi (Menge 1982). Depending on the appli- mental stations where mineral nutrients are not limiting fac- cation, fungicides applied as soil drenches are also detri- tors. Since increasing soil fertility diminishes mycorrhizal mental to AM fungi (Perrin and Plenchette 1993; Udaiyan et development, and therefore the benefits of mycorrhizae, it al. 1999). was hypothesized that this could lead to the selection of varieties with high P requirements. In other words, breeders AM Fungi Management in the Field would be selecting against mycorrhizal dependency There are two methods of managing AM fungi in the field: (Plenchette 1982). Baylis (1975) mentions that tomatoes working with indigenous AM fungi or introducing selected that do not show phosphorus deficiency have a P tissue con- strains. 34 CANADIAN JOURNAL OF PLANT SCIENCE

(i) Selected Strains with increasing fallow durations (Duponnois et al. 2001). Selected strains of AM fungi inoculum are not presently During the first fallow year, the protected fallow benefits available in large quantities at a low price. Even with sig- from a large plant biodiversity favoured AM fungi develop- nificant progress in producing AM fungi in a bioreactor ment. When the fallow was maintained for several years, (Jolicoeur et al. 1999), production will likely remain costly graminaceous plants and herbs gradually disappeared and and will be targeted at plants with high added value. Ideally, were replaced by bushes with tap roots that do not con- if AM inoculum is to be used for large crops it should not be tribute to maintaining AM fungi in the top soil. Subsequent more expensive than Rhizobium inoculum. Alternatively, crops, mainly mil and groundnut, were therefore grown in inoculum can be produced on-site under local agronomic soil that had low mycorrhizal soil infectivity. In summary, conditions (Sieverding 1991). This approach has great agricultural practices determine the detrimental or beneficial promise, but managing AM fungi in the field is difficult effects of fallow on mycorrhizal soil infectivity. under our current cropping systems. Cover crops, inserted in a cropping sequence between two The successful introduction of a foreign microorganism cash crops, are sometimes used instead of bare fallow to into the soil depends on how well it adapts, develops, and improve soil fertility and to protect the environment. Some competes for nutrients. For biological control of pests the cover crops, such as California bluebell (Phacelia sp) or introduction of a control agent is considered ecologically mustard, are used as nitrate trapping crops. Species used as successful if micro-organisms can be found in the soil sev- cover crops may also have allelopathic effects (Rizvi and eral years after inoculation. An introduction is considered Rizvi 1992); glucosinolates contained in cruciferous agronomically successful if the level of the population is residues are transformed in the soil into isothiocyanates sufficiently high to bring sizeable benefits. It is important to which act as biofumigants against weeds and fungi monitor introduced species and to determine soil receptive- (Grodzinsky 1992). Almost all plants have some allelopath- ness (Duvert et al. 1990; Plenchette 2000). On-farm select- ic effects (Rice 1974). To date mycorrhizae and allelopathy ed strains are certainly better adapted to edaphic conditions have been studied independently; the consequences of prac- than selected strains produced in vitro or in vivo under con- tices such as using mustard as a nitrate trapping crop on AM trolled conditions. Since AM fungi are obligatory sym- fungi survival have not been yet considered. bionts, their introduction can be complicated. Although soil characteristics are important, the successful introduction of COHERENCE OF SOME CURRENT CROPPING AM fungi strains depends mostly on the crop series. SYSTEMS Sebillotte (1990a) stresses that agricultural practices should (ii) Indigenous Fungi not be considered independently. Instead we should consid- Managing indigenous fungi appears to be a better option er ‘cropping systems’, which include all technical practices than inoculation. These fungi may have lower efficiency used on a field or a group of fields, such as the crop species, than selected strains, but they are well adapted to the soil. their sequence, and the management of each crop. This

For personal use only. Arbuscular mycorrhizal fungi can be effectively managed approach is vital if we are to take into account the contribu- by raising the natural mycorrhizal soil infectivity through tion of AM fungi. Cropping systems vary greatly according the use of appropriate on-farm practices. Control of fertilizer to country, climate, soil type, and even within a single farm. and pesticide applications, crop rotations that exclude non- The cropping system practiced in a field must be analyzed mycorrhizal species of Chenopodiaceae and Cruciferaceae, according to two points of view. First, from an economical reduced tillage intensity, intercropping, and cover cropping and organizational point of view, all the decisions guiding are all means to manage indigenous AM fungi. Indigenous the choice of technical aspects of crop management must be strains should be tested for efficiency. selected within a unique framework determined by the pro- Cropping sequences directly affect the abundance of AM duction goals, the constraints of the farm, and the resources fungi populations. In a long-term experiment that examined available. Farmers tend to make some of these choices based ‘Deherain’s plots’, established near Paris in 1875, it was on the current state of their land, which depends on its pre- found that crop rotation involving wheat, a low mycotroph- vious use. Second, from an ecological point of view, the dif- ic plant, and beet, a non-mycotrophic one, led to the virtual ferent technical aspects that make up a cropping system may

Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 122.56.27.149 on 11/27/18 total disappearance of AM fungi population (Plenchette influence the same soil attributes. A single practice can 1989). Fallow, a period devoted to the restoration of soil’s modify different environmental variables. For example, soil fertility, is often detrimental to AM fungi. A phenomenon, tillage affects soil porosity and the location of parasite known as long (>12 months) fallow disorder (Thompson inoculum, AM fungi, and weed populations. A single 1987), is a consequence of cultivating soil to control weeds environmental variable can also be affected by many prac- during fallow. When weeds are not controlled mycorrhizal tices. Epidemics of eye spot on wheat, for example, are infectivity is maintained, as observed by Plenchette (1989) linked to crop succession, seeding date, soil tillage, and in ‘Deherain’s plots’. In the Sahel zone, soils have tradi- cultivar. tionally been left in fallow for several years. Population In the following sections of this article we will consider increases, however, have led to shorter fallow durations. several representative cropping systems and discern the con- When the fallow was protected from cattle grazing, AM tribution of AM fungi. We will also discuss the ways in fungi populations were restored after one year of fallow. which these current cropping systems could evolve to max- The extent of AM fungi population restoration decreased imize AM fungi related benefits. PLENCHETTE ET AL. — MANAGING ARBUSCULAR MYCORRHIZAL FUNGI IN CROPPING SYSTEMS 35

Low-input Cropping Systems in Tropical Areas: In these types of low-input cropping systems, the fragili- Staple Food Production in Haiti ty of the farms and an aversion to risk are conflicting with a In developing countries of tropical humid areas, the produc- fast development of chemical inputs susceptible to allow a tion of staple foods, such as maize, sorghum, bean, cassava, consequent yield improvement. Farmers who do not use fer- and sweet potato is common. These crops are managed with tilizers work harder and increase cropping pressure on their low input levels and are intended to meet local demand. plots. Yields remain low and agricultural work is poorly Oxisols, which are highly weathered and leached soils char- rewarded. There is no doubt that these low input cropping acterized by low CEC and low nutrient availability, are systems are not the choice of farmers to preserve the envi- dominant (Engelstad and Russel 1977). Risks of aluminic ronment. On the contrary, overuse of soils at the lowest pos- toxicity are high and soil P fixing power is large. Thus, crop sible cost leads to soil fertility degradation and lower yields. mineral nutrition is often the primary factor in limiting And what is the current mycorrhizal infectivity of these yield. The cropping systems of upland Haiti are one exam- soils? The absence of chemical inputs might suggest that the ple of the low input cropping systems that are typical of the soil conditions would be favourable for AM fungi. tropics. However, in comparable low-input systems in Madagascar, In Haiti, agriculture is not mechanized. The dominant the mycorrhizal development of current crops or plants crop, which receives neither pesticides nor chemical fertiliz- (potatoes, peas, corn, crotalaria, brachiara) were very limit- ers, is a maize-bean intercrop into which root crops (usually ed or not observed (Plenchette, unpublished data). This sit- sweet potato) are sometimes added. Bean and maize are uation can be attributed to several factors, including: the sown on the same day, either in February or in July, and har- global deficiency of mineral nutriments, the suppression of vested 2 months later for bean, and 6 months later for maize fallow (loss of biodiversity), and practices such as burning and sweet potato. In most fields, periods of cultivation are off crop residues. Is the inoculation of selected strains of separated by a natural grazed fallow. Increasing demo- AM fungi possible? Clearly, the answer depends on the graphic pressure, however, has led to shorter fallow periods unique conditions of each situation. For instance, according that typically do not exceed 8 months. This reduction in fal- to the mycotrophic behaviour of species, the introduction of low period length is associated with reductions in rearing, cabbage (a non-mycotrophic crop) in rotation with the bean- manure production, and yields. Continuous cropping on maize intercrop could be less favorable than continuous sin- steep slopes has favoured soil erosion. Today, yields are gle intercrop practised over the years. In natural grassland, often lower than 500 kg ha–1 for bean, 600 kg ha–1 for AM fungi were found to play a role in determining the com- maize, and 1000 kg ha–1 for sweet potato. An agronomic position and dynamics of the plant community (Allen et al. diagnosis shows that limiting factors of yield are low nutri- 2002). Conversely, multiple cropping systems (mixed crop- ent availability, bean root fungal diseases that become more ping, intercropping, alley cropping, relay intercropping) that severe with shorter intervals between bean cultivations, and produce two or more crops in the same field each year may fall armyworm damage (Spodoptera frugiperda) on maize help to maintain a high level of AM fungi biodiversity (Hart

For personal use only. from insects emerging from maize crop residues of recently and Klironomos 2002); monocultures tend to select one harvested fields (Clermont-Dauphin 1995; Clermont- species which is not necessarily the most efficient one Dauphin et al. 2003). (Johnson et al. 1992). With these concurrent decreases in cropped land and crop productivity, farmers are growing poorer. The introduction High-input Cropping Systems in Tropical Areas: of high input management is not feasible under these condi- Banana Monoculture in the French West Indies tions and is limited to cash crops, such as market garden In tropical areas, high input cropping systems are generally crops (cabbage, potatoes) that are sometimes introduced in maintained for export production. Problems of sustainabili- rotation with the bean-maize intercrop. In trying to reduce ty, although similar to those in other temperate areas, can be risk, farmers may make technical choices that compromise exacerbated because: (i) higher amounts of fertilizer are the efficiency of their chemical inputs. For example, farm- used to meet potentially higher crop requirements; (ii) high- ers may choose varieties based on their tolerance to para- er pesticide applications are needed to counter the higher sitism and low soil phosphorus availability regardless of parasite pressure which is linked to relatively uniform year-

Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 122.56.27.149 on 11/27/18 yield potential. Another example is maintaining intercrops round temperature regimes, and; (iii) more rainfall and instead of pure crops even though competition from bean steeply sloped land increase the risks of soil degradation and strongly limits maize productivity. The intercrop has the the leaching of nutrients. The banana cropping systems of advantage of ensuring a more stable level of production; low the French West Indies are typical of the kind of high-input bean yield could be compensated by higher maize yield in cropping systems that are common in the tropics. the same plot. Another advantage to farmers of intercrop- Banana production is mainly localized in areas where ping is that maize production does not affect the main bean banana is the only crop. Plots are replanted every three to crop. If a low maize grain yield is probable, the maize crop five years; repeated passes with heavy machinery destroy can be turned into forage for the herd as soon as the bean is and bury preceding banana plant residues and prepare the harvested. The bean stand is composed of a mixture of soil. Chemical inputs are abundant and sometimes exceed genotypes; some are highly productive and others show crop needs. For example, annual applications of 400–600 kg some resistance to low P availability of soils and soil-borne ha–1 of N and 400 kg ha–1 of K are common even though the diseases. exports of the harvested fruit do not reach 80 kg ha–1 for N 36 CANADIAN JOURNAL OF PLANT SCIENCE

and 200 kg ha–1 for K. Three applications of nematicide/ mental damage. In the northern half of France many farms insecticides, corresponding to about 20 kg ha–1 of active have abandoned livestock production in favour of field ingredients, and about four herbicide applications are made crops over the last 20 to 40 years (Sebillotte 1990b). Winter every year. This high reliance on cropping inputs is justified wheat, which is the main species cultivated on these farms, by the farmer’s primary objective of high yields. The mar- is grown in rotations that often include barley, sugar beet, ket requirements and the need to make the organization of oilseed rape, fodder pea, maize, potatoes, and sunflower. the work easier also justify these high-input cropping sys- The relatively small size of the farms and the high price of tems. To obtain quality fruit of consistent size, high and sus- farm products on the internal market (at least until recent tained nutrient uptake must be ensured during the cropping years) have led farmers to adopt intensive crop management period. This is achieved through frequent fertilizer applica- systems that aim at maximizing yield (Meynard and tions and regular pest control treatments. Replanting allows Girardin 1992). Yields of winter wheat produced on good for harvest dates to be better synchronized and makes it eas- soils often exceed 8 tons per hectare. To maximize yields, ier to circle the plantation. Nematode-free tissue cultured farmers try to maximize the interception of radiant energy; plants are used for replanting to decrease pressure from the seeding is scheduled in early autumn and seeding densities nematode Radopholus similis, at least during the first year, have increased in the past 20 years, thus encouraging pests provided the field has been disinfected by a previous fallow and fungal diseases. To counter these risks and secure high (Chabrier and Quenehervé 2003). Ploughing to bury the yields, farmers generally apply preventative fungicidal residues of the past plantation can lead to soil structure treatments (one to three according to the year and region), degradation and watterlogging (Perret and Dorel 2001). insecticidal treatments in the autumn and sometimes in the This practice is also associated with a decrease in soil organ- spring, and high levels of nitrogen fertilizer split into sever- ic carbon, microbial respiration, and earthworm biomass. al applications to match crop demands. The environmental damage resulting from such cropping systems has been well Frequent nematicide applications disturb the soil biotic documented and includes the pollution of groundwater by equilibrium, making it more succeptible to R. similis infes- nitrates and pesticides and a reduction of biodiversity in the tations (Clermont-Dauphin et al. in press). Thus, the use of cereal-growing plains. Various studies have shown the envi- nematicides can foster dependency. Anecdotal information ronmental benefits of using predictive models to calculate from farmers supports this research; they suggest that the fertilizer applications and pesticides treatments, of sowing control of R.similis has required greater and more frequent catch crops to absorb nitrates and to prevent winter leaching, nematicide applications over the years. They also report that and the use of disease-resistant varieties (Rossing et al. yields tend to decrease while input levels have been 1997; Meynard et al. 2002). increasing. High input cropping systems include: (i) non-mycotroph- High-input cropping systems in tropical areas are not ic crops of oilseed rape, sugar beet, and mustard as a catch favorable for mycorrhizal development; high levels of fer- crop; (ii) low-mycotrophic plants of wheat and barley; (iii) tilizer, massive applications of fertilizer, and large inter- For personal use only. moderate-mycotrophic crops of maize and sunflower, and; rows of bare soil that are regularly weeded are detrimental (iv) highly-mycotrophic crops of potato and pea. The myc- to AM fungi (Declerck et al. 1995b). Soil receptiveness to orrhizal state of the soils can vary greatly according to the mycorrhizal fungi was found to be negatively correlated agricultural region and the choice of crop rotation. For with soil P content (Plenchette 2000). This rather dark prog- example, in Picardy and in the chalky Champagne country, nosis, however, does provide some insights into how to new local processing industries and the high proportion of build more sustainable cropping systems. Practices to be deep soils with large water-holding capacities allow farmers considered include: suppressing tillage to reduce the risks of to grow potatoes, peas, and beans for processing, and alfal- parasite development; improving biological interactions to fa, which is converted to stockfeed for other regions. In this control pathogens, and; adjusting fertilization for soil type to case, all plants are generally highly mycotrophic and the minimize environmental risks. The sowing of cover crops cropping system supports a high level of mycorrhizal soil between rows of banana, possibly controlled by hand weed- infectivity. Conditions are favourable for AM fungi, espe- ing, could help to limit erosion and increase biological activ- cially when farmers practise IPM (integrated pest manage- ity in the soil, particularly that of AM fungi and earthworm. Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 122.56.27.149 on 11/27/18 ment) and have reduced fungicidal treatments, provided the At least under controlled conditions, some varieties of bioavailable P content of the soil is not too high. In contrast, banana exhibit a high mycorrhizal dependency (Declerck et in regions with dry summers and shallow soils, which are al. 1995a). Interactions between AM fungi and Cylindro- unfavourable to crops harvested at the end of summer (pota- cladium spathiphylli (Declerck et al. 2002) and R. similis to, sunflower, maize, sugar beet, etc.), rotations include (Elsen et al. 2001) decrease both the populations of mainly cereals and oilseed rape, which are inimical to myc- pathogens and disease severity in banana plants. These orrhiza. The use of mustard as a catch crop to limit the risk recent results should be confirmed by field experiments. of nitrate leaching from the surface soil probably aggravates the situation. The prevalence of wheat crops makes it more High-input Farming Systems in Temperate difficult to reduce fungicide treatments. Over time, rotations Regions: Cereal Systems in France including non-mycotrophic or low-mycotrophic crops and Cereal systems in northwestern Europe are also fairly inten- associated pesticide treatments lead to a decrease in mycor- sive, although they are being adapted to reduce environ- rhizal soil infectivity (Plenchette 1989). The more frequent- PLENCHETTE ET AL. — MANAGING ARBUSCULAR MYCORRHIZAL FUNGI IN CROPPING SYSTEMS 37

ly these crops are grown, the greater the decrease in mycor- AM fungi. The benefits of mycorrhizae are undeniable and rhizal soil infectivity. In this case species diversification may still be underestimated due to the difficulties of charac- would reduce plant parasitism, provide weed control with terising mycorrhizal soil infectivity; mycorrhizal soil infec- less herbicide use, and increase the efficiency of mycor- tivity is rarely quantified in agronomic studies. rhizae. However, in an open international market, the pric- In order to take advantage of mycorrhizae through innov- ing of agricultural products makes diversification ative cropping systems, the following three conditions must improbable; environmental protection is likely to come only be met: as a result of government incentives. (i) We need quality indicators of AM fungi population and Intensive Livestock Systems in Temperate AM development on plants. We need to be able to charac- Regions: Pig and Cattle Farms in Western France terize AM fungi and mycorrhizae as easily as soil richness Just as some French regions specialize in crop production, in terms of exchangeable cations. This may permit AM others specialize in animal production. In the west of fungi characterization to be integrated with an agronomic France, production is intensive (industrial pig and poultry diagnosis approach (Doré et al. 1997). This would help us to units, high production dairy cattle); the concentration of discern when low mycorrhizal development leads to low local processing industries and technical advances have con- production. tributed to herds that far exceed the numbers that could be (ii) We need a better working knowledge of the relationship fed from local crop production. In these intense livestock between cropping systems and AM fungi so that myc- systems, dairy cows no longer graze on natural pastures but corhizae can be integrated into crop models. Innovations in eat ryegrass and maize silage which is balanced with pro- cropping systems will rely more and more on scenarios pre- teins from soybean cake. Pigs eat cereals (wheat or barley) dicted by models (Meynard 1998, 2001; Boiffin et al. 2001); that are produced locally but also receive cereals, protein experimentations “in silico” will allow us to predict the con- peas, and soybean from other regions or countries. These sequences of different combinations of cropping practices imports lead to considerable positive mineral balances at the under various climates. farm level, which contribute to nitrate pollution of surface and ground water, and sometimes to extreme P enrichment (iii) We need economical and social conditions that promote of soils (Pujol and Dron 1998). Even when favourable crops sustainable production instead of short-term solutions that occupy an important place in the rotation, this P enrichment meet immediate needs. Considerations for a better valoriza- is detrimental to AM fungal development. In some systems tion of mycorrhizae, and the current deadlock of some of our of dairy cattle or pig production, AM fungi have complete- cropping systems, call into question the economical logic ly disappeared. The bioavailable P content of the soils is that has led farmers to shorten their fallows and/or saturate often so high that it would require about 10 years without their soil with phosphorus. The separation between cropping fertilizer applications to soak up the excess P and permit the and intensive animal production, sometimes out of ground

For personal use only. reappearance of AM fungi. In Britanny, a great region of in some part of the world, shows its limits, and the use of soil-less culture (poultry, pork), excess P in some soils is pesticides as the preferential means of controlling the such that no changes in soil P status are expected for some ecosystem, appear to constitute a no-win game of sorcerer’s hundreds of years; on one poultry farm P levels were mea- apprentice. sured at 7000 µg g–1 (using Dyer’s method). Under these Sustainable, innovative cropping systems should favour conditions, improvements in mycorrhizal symbiosis are next beneficial microbial interactions, in particular, those affect- to impossible. ing plant nutrition and protection. No-till, low input crop- Phosphorus is more often sorbed in soils (Barber 1984); ping systems with permanent cover crops, either dead or mycorrhizal development is governed by the P concentra- alive, have been successfully tested over several years in tion in the soil solution (Habte and Manjunath 1987). With Brazil and Madagascar on extremely poor soils. These sys- a high level of AM inoculum, mycorrhizae can be efficient tems prevent soil erosion, preserve organic matter, and in high P soils, provided they have a high fixing power that increase microbial biomass. Soil fertility is recovered with- limits the exchanges between the sorbed P and the soil solu- out high fertilizer inputs (Séguy and Bouzinac 1998; Séguy

Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 122.56.27.149 on 11/27/18 tion (Plenchette and Fardeau 1988). If AM fungi have dis- et al. 1998) and the soils seem highly favourable to AM appeared for reasons other than fertilization, it is difficult to fungi and mycorrhizal development. Some of these systems prescribe a yearly inoculation of AM fungi even in the case have also been tested in France (Ghiloufi et al. 2001). of high P-fixing soils. Clearly we have seen that some simplified and highly specialized systems have led to irreversible situations where CONCLUSION soil P levels preclude the introduction and maintenance of Clearly, the best situation for AM fungi maintenance is a AM fungi for hundreds of years. New lower-input (but not cropping system in which all components have positive zero external input) agro-ecosytems will become more effects on AM sysmbiosis. The worst conditions for AM sophisticated and complex. New methodologies should be fungi maintenance include: high soil P fertility, cropping developed to predict the behaviour of AM fungi under the systems involving non-mycotrophic crops, and the use of constraints of cropping systems, and models of mycorrhizae fungicides. The four examples presented here clearly show development should be tested in long term field experi- that current cropping systems are severely detrimental to ments. Maintaining the population of AM fungi as the 38 CANADIAN JOURNAL OF PLANT SCIENCE

essential link between the soil and plant is an essential con- orrhiza on plant growth. II. Influence of soluble phosphate on dition of sustainable cropping systems. endophyte and host in maize. New Phytol. 68: 945–952. Declerck, S., Plenchette, C. and Strullu, D. G. 1995a. Adjoud, D., Plenchette, C., Halli-Hargas, R. and Lapeyrie, F. Mycorrhizal dependency of banana (Musa acuminate, AAA 1996. Response of 11 eucalyptus species to inoculation with three group) cultivar. Plant Soil, 176: 183–187. arbuscular mycorrhizal fungi. Mycorrhiza, 6: 129–135. Declerck, S., Plenchette, C., Risede, J. M, Strullu, D. G. and Allen, M. F. and Boosalis, M. G. 1983. Effects of two species of Delvaux, B. 1995b. Estimation of the population density of arbus- VA mycorrhizal fungi on drought tolerance of winter wheat. New cular mycorrhizal fungi in soils used for intensive banana cultiva- phytol. 93: 67–76. tion in Martinique. Fruits, 54: 3–9. Allen, M. F., Lansing, J. and Allen, E. B. 2002. The role of myc- Declerck, S., Risede, J. M., Rufyikiri, G. and Delvaux, B. 2002. orrhizal fungi in the composition and dynamics of plant communi- Effects of arbuscular mycorrhizal fungi on severity of root rot of Cylindrocladium spathiphylli 51 ties: A scaling issue. Pages 344–367 in K. Esser, U. Luttge, W. bananas caused by Plant Pathol. : 109. Beyschlag, and F. Hellwig, eds. Progress in botany. 63. Springer- Doré, T., Sebillotte, M. and Meynard, J. M. 1997. A diagnostic Verlag Berlin. method for assessing regional variations in crop yield. Agric. Syst. Alexandratos, N. 2003. World agriculture towards 2015/2030. 54 Proc. IFA-FAO agriculture conference: Global food security and : 169–188. the role of sustainable fertilization. Rome, Italy. Dorneless, M. R. F., da Silva, C. M and Gomes, A. A. 2001. A Arihara, J. and Karasawa, T. 2000. Effect of previous crops on model for hyphae effects in phosphorus absorption by plants. arbuscular mycorrhizal formation and growth of succeeding maize. Ecological Modelling 142: 83–89. Soil Sci. Plant Nutr. 46: 43–51. Duponnois, R., Plenchette, C. and Bâ, A. 2001a. Growth stimu- Azcon, R. and Ocampo, J. A. 1981. Factors affecting the vesicu- lation of seventeen fallow leguminous plants inoculated with lar-arbuscular infection and mycorrhizal dependency of thirteen Glomus aggregatum in Senegal. Eur. J. Soil Biol. 37: 181–186. wheat cultivars. New Phytol. 87: 677–685. Duponnois, R., Plenchette, C., Thioulouse, J. and Cadet, P. Baltruschat, H. and Schönbeck, F. 1972. The influence of 2001b. The mycorrhizal soil infectivity and arbuscular mycor- endotrophic mycorrhiza on the infestation of tobacco by rhizal fungal spore communities in soils of different aged fallows Thielaviopsis basicola. Phytopathol. Z. 84: 358–361. in Senegal. Appl. Soil Ecol. 17: 239–251. Barber, S. A. 1984. Soil nutrient bioavailability. A mechanistic Duvert, P., Perrin, R. and Plenchette, C. 1990. Soil receptive- approach. John Wiley and Sons Inc., New York, NY. 398 pp. ness to VA mycorrhizal association. Concept and method. Plant Baylis, G. T. S. 1959. Effect of vesicular-arbuscular mycorrhizas Soil 124: 1–6. on growth of Grielinia littoralis (Cornaceae). New Phytol. 58: Edwards, C. A., Madden R. L. P., Miller, R. H. and Hause, G. 274–280. 1990. Sustainable agricultural systems. Soil and water conserva- Baylis, G. T. S. 1975. The magnolioid mycorrhiza and mycotro- tion society, 7515 Nortyhesat Ankeny Road, Iowa, USA. phy in root systems derived from it. Pages 373–389 in F. E. Elsen, A., Declerck, S. and De Waele D. 2001. Effects of Glomus Sanders, B. Mosse and P. B. Tinker, eds. Endomycorrhizas. intraradices on the reproduction of the burrowing nematode Academic Press, London and New York. Radopholus similis in dixenic culture. Mycorrhiza Bowen, G. D. 1975. Endogone strain and host plant differences in Engelstad, O. P. and Russel, D. A. 1977. Fertilizers for use under development of vesicular-arbuscular mycorrhizas. Pages 77–86 in tropical conditions. Adv. Agro. 27: 175–208.

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PERSPECTIVE

Fungal inoculants in the field: Is the reward greater than the risk?

Miranda M. Hart1 | Pedro M. Antunes2 | Veer Bala Chaudhary3 | Lynette K. Abbott4

1University of British Columbia Okanagan, Kelowna, BC, Canada Abstract 2Algoma University, Sault Ste. Marie, ON, 1. Biofertilizers are a large part of the global agricultural economy. Canada 2. Recently, there has been an increase in the number of companies producing fungal 3DePaul University, Chicago, IL, USA inoculants. 4University of Western Australia, Crawley, WA, Australia 3. Whether these inoculants are useful is not clear; they are difficult to monitor in the field. Correspondence Miranda M. Hart 4. The unintended consequences of inoculants in natural systems is not known, but if Email: [email protected] invasive, they may pose a threat to soil and plant biodiversity and ecosystem

Funding information functioning. NSERC, Grant/Award Number: Discovery Grant; Gledden Foundation KEYWORDS arbuscular mycorrhizal fungi, biofertilizers, fungal inoculants, invasive fungi, sustainable Handling Editor: Katie Field agriculture

1 | INTRODUCTION reduce inputs. These claims are based on the growing body of knowledge clearly showing that AMF help plants survive stressful growing There is a global industry built upon the production of “bioinoculants,” conditions (Lenoir, Fontaine, & Lounès-Hadj, 2016; Liu et al., 2007). which includes arbuscular mycorrhizal fungi (AMF). These fungi es- However, unlike other bioinoculants (i.e. rhizobia), there is yet little tablish symbiotic associations with the roots of most plants and are proof that inoculation by commercial AMF is useful in cropping sys- known to improve plant performance. Currently, only a few AMF tems. Are increases in yield sufficient to offset the added expense of genotypes are produced as bioinoculants and they are distributed inoculum application? Are the results consistent/predictable? While globally, but evidence for their efficacy is scant, incomplete or lacking the idea of increasing agricultural yield without concomitant increases altogether. Perhaps more troubling is that the scientific community in fertilizer is appealing environmentally, there are many unknowns has little idea of the ecological consequences of the use of AMF inoc- about these products. ula. This is a clear case where industrial practices are not aligned with Despite the reported variability in plant growth promotion fol- current scientific knowledge. The ethical and economic implications of lowing inoculation with AMF (Hoeksema et al., 2010), a growing such oversight are profound, and ranges from customer quality assur- number of companies world-wide produce and market AMF inoc- ance to soil biodiversity and ecosystem functioning. Here, we outline ulants (Dalpé & Monreal, 2004) (Figure 1). There is no universally important gaps in knowledge, both in terms of inoculant efficacy and adopted code of “best practice” regarding AMF inoculum selection ecosystem integrity. and quality control. Perhaps the research community has failed to effectively communicate the lack of evidence for guaranteed benefits or perhaps the prospects of commercial gains may have 2 | A RECURRING REVOLUTION spiked the optimism of inoculant producers. Whatever the reason, the burgeoning inoculant market has flourished in the absence of Every 10 years or so, the idea of using AMF as a bioinoculant is re- adequate risk assessment or global regulatory policies on their pro- discovered (e.g. Abbott & Robson, 1982; Hart & Trevors, 2005; duction and distribution (Malusá & Vassilev, 2014; Owen, Williams, Mcgonigle, 1988; Rodriguez & Sanders, 2015). The story is similar in Griffith, & Withers, 2015; Vosátka, Látr, Gianinazzi, & Albrechtová, every iteration: AMF are powerful tools that will improve yields and 2012).

(a) (b) (c)

FIGURE 1 Studies examining arbuscular mycorrhizal fungi (AMF) bioinoculants conducted by country from 1970 to 1999. n = 193 (a), and 2000 to 2014, n = 444 (b). Inoculum producers by country that cited in both (a) and (b). Countries producing inoculum that were cited in (a) and (b), n = 43 (c). We searched Web of Science (Thomson Reuters) on 6 August 2015 using Topic: (Mycorrhiza*) AND Title: (Inocul*) Time span: 1970–2014 which returned 1,350 papers total, 1,286 in English. We then excluded all papers not dealing with AMF and/or inoculum application. In total, we surveyed a total of 631 papers. A list of all companies is included in Table S1

3 | HARD TO EVALUATE OUTCOMES yield or other variables that correspond to meaningful gains in cropping systems. If previous attempts to incorporate AMF inoculants into mainstream agrosystems were stalled by lack of research tools (Hart & Trevors, 4.1 | Partner identity 2005; Schwartz et al., 2006), this is no longer an obstacle. Molecular advances in the last decade now make it possible to track inocu- Variation in host response to inoculation may be partly explained by lation of single genotypes (Farmer et al., 2007; Hart et al., 2013; the identity of the host and fungus. For example, host response to Jansa, Smith, & Smith, 2008; Rodriguez & Sanders, 2015). However, inoculation can vary with cultivar (Buysens et al., 2016; Ceballos et al., despite the ability to assess the abundance of inoculants in the en- 2013; Douds et al., 2016; Pellegrino et al., 2015). Similarly, fungal vironment, the effectiveness and risk of AMF inoculation remain provenance can lead to different inoculation outcomes: positive in- unclear. oculation effects are associated with specific fungal isolates (Abbott & Robson, 1982; Abbott et al., 1983), while other studies show positive host response to inoculation when using local fungal inoculants 4 | EVIDENCE FOR IMPROVED (Caravaca et al., 2005; Davidson, Novak, & Serpe, 2016; Emam, 2016; PLANT RESPONSE Labidi et al., 2015; Maltz & Treseder, 2015; Middleton et al., 2015; Pellegrino & Bedini, 2014; Rúa et al., 2016). Given that the efficacy of Meta-analyses indicate that growth responses to AMF inoculation the AM symbiosis is influenced by soil fertility (Tawaraya et al., 2012), are, on the whole, inconsistent (Hoeksema et al., 2010; Lekberg & inoculation timing (Mummey, Antunes, & Rillig, 2009), site distur- Koide, 2005; Mcgonigle, 1988; Pellegrino, Öpik, Bonari, & Ercoli, bance level (Antunes et al., 2009) and, perhaps partner co-adaptation 2015). Part of the reason for inconsistency within these analyses (Koyama, Pietrangelo, Sanderson, & Antunes, 2017; Rúa et al., 2016), stems from the fact that both field and greenhouse studies were it is not surprising that it is difficult to detect a uniform host response considered together, but high variation persists even among field to inoculation. This does not bode well for predictable commercial in- studies. A spate of recent studies show an increase in plant bio- oculation outcomes. mass post inoculation (e.g. Camprubi, Zárate, Adholeya, Lovato, & Calvet, 2015; Cely et al., 2016; Hijri, 2016; Pellegrino et al., 2012; 4.2 | Inflated positive outcomes Tawaraya, Hirose, & Wagatsuma, 2012). Most of these studies, especially those evaluating multiple plant taxa, either show posi- Is the surfeit of recent, positive outcomes applicable to real world sce- tive responses to inoculation (e.g. Abbott & Robson, 1982; Abbott, narios? Most inoculation studies are conducted under artificial green- Robson, & Hall, 1983; Antunes et al., 2009; Buysens, César, Ferrais, house conditions which do not inform performance in the field (Faye Dupré de Boulois, & Declerck, 2016; Caravaca, Alguacil, Barea, et al., 2013; Ohsowski, Zaitsoff, Öpik, & Hart, 2014). Furthermore, & Roldán, 2005; Ceballos et al., 2013; Douds, Wilson, Seidel, & biases within field studies may be more pernicious, as they are more Ziegler-Ulsh, 2016; Emam, 2016; Middleton et al., 2015) or no subtle than those in greenhouse studies. effect at all (e.g. Farmer et al., 2007; Pellegrino & Bedini, 2014). Studies showing reductions in plant performance following in- 4.2.1 | Degraded systems oculation have also been reported (e.g. Aprahamian et al., 2016; Herzberger, Meiners, Towey, Butts, & Armstrong, 2015; Janoušková The majority of field inoculation studies have been conducted in et al., 2013; Verbruggen, Kiers, Bakelaar, Röling, & van der Heijden, severely degraded soils (i.e. restoration post mining) or soils with 2012). However, few studies measure plant reproductive output, very low inoculum potential (e.g. some situations in conventional 128 | Functional Ecology HART et al. agriculture or desertified landscapes). Extreme conditions increase horticultural crops (i.e. grapevines and some field vegetables), the probability of detecting a positive inoculation outcome because in general, the existence of AMF-free plants is highly unlikely propagule abundance is tightly linked to mycorrhizal response (Allen (Lekberg & Koide, 2014). Thus, using uninoculated “controls” in & Allen, 2005). Thus, AM plants should respond positively to inocu- systems requiring transplants does not represent a valid test of lation when growing in an inoculum-limited environment (Abbott & inoculation response. Robson, 1982). While studies in degraded systems are a useful indication of commercial inoculant performance in extreme conditions, they cannot inform inoculant use in many other systems, where resi- 5 | EVIDENCE FOR SUCCESSFUL dent AMF communities are well-established, as in most agricultural ESTABLISHMENT? soils (Oehl et al., 2010). Furthermore, custom inocula sourced from reference ecosystems have been shown to perform better than com- In most field studies, inoculant establishment is not reported. mercial inocula in restoration campaigns (Maltz & Treseder, 2015). To Without evidence of inoculant establishment, responses are merely balance knowledge of inoculant performance, it will be important to correlative, and it is not possible to ascribe changes in host perfor- conduct inoculation experiments across a broad range of soil condi- mance to the introduction of AMF inocula (Rodriguez & Sanders, tions, including different land use types, and intact ecosystems to 2015). If inoculation results in a growth response in the absence better predict where inoculation may be effective. Understanding the of inoculant establishment, can we say that inoculation is “success- local conditions is essential for predicting success of inoculation as the ful?” Unless carefully controlled for, observed changes (positive or absence of AMF may reflect unsuitable soil conditions or land man- negative) maybe due to concurrent “fertilizer/amendment effects” agement practices that may preclude establishment and persistence potentially associated with inoculants, such as the co-introduction of inoculant AMF. of other microbiota with the inoculant carrier, inclusion of nutrients or simply due to inoculation protocols, rather than the AMF in the inoculant itself. 4.2.2 | Inappropriate controls

Erroneous comparisons can result when inoculated plants 5.1 | Root colonization are compared to non-inoculated controls (Boyer, Brain, Xu, & Jeffries, 2015; Burkle & Belote, 2015). For example, where plants While root colonization is commonly reported in studies of responses must first be propagated in nurseries prior field transplantation, to inoculant AMF, this information reflects inoculant establishment uninoculated “controls” typically perform more poorly than in- only in systems with no resident fungi. It has little value in field tri- oculated plants (e.g. Camprubi et al., 2015; Hernádi, Sasvári, als because even in extremely degraded soils, it is difficult to distin- Albrechtová, Vosátka, & Posta, 2012; Tawaraya et al., 2012). In guish among inoculants and fungi naturally dispersing into the site this situation, control plants are deprived of the AM symbiosis (Bell, Wells, Jasper, & Abbott, 2003). In such conditions, it can at best during critical developmental stages, and do not provide a valid indicate an indirect influence, such as through changes in the AMF control. While this approach may be relevant for transplanted community (Rodriguez & Sanders, 2015).

FIGURE 2 Predicting the fate of an arbuscular mycorrhizal fungi (AMF) inoculant in natural systems. Both the utility of AMF inoculants and their threat as invasive species depend on their ability to complete their life cycle in a novel environment. There are multitude barriers which can interfere with their ability to establish, colonize, persist and spread HART et al. Functional Ecolo gy | 129

Box 1 What determines if an inoculant will spread? (Associated with Figure 2)

Successful AMF establishment is a complex process involving biotic and abiotic interactions. As host requirements vary both temporally and spatially (e.g. nutritional, pathogen protection), conditions that make an AMF more successful/beneficial will not be constant over time or space. Germination

1. Propagule type: Inoculation will fail if it does not contain the appropriate propagule. Not all AMF use the same propagule (i.e. spores/hyphae/root fragment). While this may have a taxonomic basis, information on most taxa is lacking (Klironomos & Hart, 2002). 2. Germination requirements: Germination has specific abiotic/biotic triggers. Soils with incompatible conditions may fail to trigger germination, and spores may remain quiescent until they degrade or are depredated. Similarly, propagules with strict dormancy requirements may fail to establish in an appropriate time frame, despite favourable conditions (Varga, Finozzi, Vestberg, & Kytöviita, 2015). 3. Viability of inoculants: Inoculum quality is a major constraint on germination. Commercial inoculants are often non-viable (Vosátka et al., 2012) and/or contain different taxa from those advertised (Faye et al., 2013). These taxa may include soil biota other than AMF.

Colonization

1. Fungal diversity: The more AMF isolates contained in an inoculant, the greater the chance they have to establish and colonize roots. However, the risk of including invasive AMF isolates may also increase with the inclusion of more isolates. 2. Dosage: Without sufficient inoculum density (i.e. propagule pressure), colonization will either fail or be functionally ineffective (Allen & Allen, 2005). Conversely, inoculation in excess of the amount needed for full colonization will be a waste of resources and may pose a risk of unintended invasion. Best practises for inoculation protocols are lacking and there is little evidence for how much, or how often, inoculants should be applied (Herrmann & Lesueur, 2013; Owen et al., 2015). 3. Order of arrival: There is increasing evidence that priority effects are important for AMF community assembly (Davison 2015; Mummey et al., 2009; Werner & Kiers, 2015). While some studies show inoculant establishment regardless of other fungi in the roots (i.e. Abbott & Robson, 1984; Hepper, 1979; Vierhillig, Lerat, & Piche, 2003), others show that resident fungi may occlude inoculants (Khaosaad, García- Garrido, Steinkellner, & Vierheilig, 2007; Vierheilig, Steinkellner, Khaosaad, & Garcia-Garrido, 2008; Werner & Kiers, 2015). 4. Niche requirements: Much has been written on the effect of soil chemistry on AMF, including differential pH tolerance (Porter, Robson, & Abbott, 1987), local adaptation to soil and hosts (Johnson, Wilson, Bowker, Wilson, & Miller, 2010), and response to salinity (Juniper & Abbott, 1993). In addition, host identity may be a major determinant of inoculation outcome (Hoeksema et al., 2010; Lekberg & Koide, 2005).

Persistence

1. Niche overlap: The ability to compete with resident fungi will determine the inoculant’s relative abundance within a mixed AMF community (Abbott et al., 1983; Jansa et al., 2008). An inoculant may fail depending on the degree of niche overlap with resident fungi. For AMF, there is evidence for reduced competition among closely related species (Engelmoer, Behm, & Toby, 2014; Thonar, Frossard, Šmilauer, & Jansa, 2014) and isolates (Roger, Colard, Angelard, & Sanders, 2013) but others show increased competition (Hart et al., 2013; Maherali & Klironomos, 2012). Other studies indicate a range of factors determine competition outcomes, including infectivity of the inoculum (Abbott & Robson, 1984; Janoušková et al., 2013), fungal life history (Abbott et al., 1983), carbon limitation (Knegt et al., 2014), drought stress (Boyer et al., 2015) and time (Wilson & Trinick, 1983). 2. Access to host carbon: Relative abundance of inoculants in the root will be determined largely by their ability to access the host’s carbon stores. If the inoculant is a superior competitor, it may dominate in host roots (Bever, Richardson, Lawrence, Holmes, & Watson, 2009; Pearson & Schweiger, 1994). Alternatively, inoculant abundance may not be related to competitive differences, and success may relate to its mutualistic ability (Fitter, 2005; Kiers et al., 2011). However, from a practical point of view, this is not predictable. There is no such thing as a superior AMF competitor or mutualist devoid of context, given variation in benefits AMF provide. 3. Fluctuating conditions: As with previous stages (germination and colonization), an inoculant will fail to complete its life cycle if conditions are outside the range of tolerance (i.e. soil nutrients, disturbance levels). Changing conditions mean that inoculants may become less suitable over time. Thus, an inoculant may for example establish prior to fertilizer application, but fail to grow once conditions change. 4. Genetic diversity: AMF used in commercial inoculants may have gone through a bottleneck and have substantially less genetic diversity than natural AMF communities.

Spread

1. Persistence: If an inoculant is able to complete its life cycle in a novel environment, it may persist, but there are no long-term studies to know how long this might be. Thus, best practises for inoculation frequency are many years away. 2. Invasion: If an inoculant is able to disperse beyond its intended zone, then it may become invasive. Information about AMF dispersal is scarce, but we do know that they can move via sediment (Harner, Piotrowski, Lekberg, Stanford, & Rillig, 2009), the atmosphere (Egan, Li, & Klironomos, 2014) and even with migratory birds (Nielsen, Kjøller, Bruun, Schnoor, & Rosendahl, 2016) and that viable propagules can passively colonize disturbed soil in a matter of weeks (Johnson & McGraw, 1988). Thus, inoculants have the capacity to be transported far beyond their intended location. 130 | Functional Ecology HART et al.

How much inoculum is necessary Which types of propagules are and how often should it be Do inoculant AMF alter microbial community structure? most effective for each isolate? applied? Do inoculant AMF exchange Are there time/abiotic triggers for What are the biotic and abiotic germination? genetic information with resident qualifiers? isolates? How pervasive is dormancy? Is it Do different isolates have context specific? Do inoculant AMF alter plant different host/biotic/abiotic communities? How long do propagules live? tolerances? Does carrier matter? How do AMF disperse? Does it Should we tailor inoculants to depend on climate/land use? How should inoculants be specific plants or soil types? stored? Can the dispersal of AMF be Should plants be pre-inoculated controlled? before planting?

FIGURE 3 Knowledge gaps and future research priorities for arbuscular mycorrhizal fungi (AMF) inoculants

So what can we say about inoculant establishment? (1) Early es- 5.2 | Tracking specific inoculants tablishment appears to be highly variable and (2) there is little evi- Attempts to directly track inoculants are few, thus knowledge of dence that inoculants persist. Considering that, on average, only 10% inoculum establishment and persistence is scant. High variability re- of all exotic species are able to establish and persist in a novel habitat ported in inoculum detection may be due to the inability of current (Williamson, 1996; also, see Figure 2 and Box 1 for What drives in- marker genes to effectively distinguish among closely related taxa. oculant spread?), it may be difficult to select an AMF inoculant that This is particularly the case for commercial inoculants, which comprise persists between growing seasons. Whether this is a concern for end cosmopolitan taxa, as the current barcoding gaps in ITS, LSU and 18S users is not yet clear. It may not be necessary for inoculants to persist are unable to distinguish among isolates within a species (Hart et al., in the soil if inoculation benefits can be achieved by repeated inocula- 2015). Approaches based on isolate-specific targets offer greater tion. However, knowledge of both the establishment and persistence promise but are less common. Again, studies using this approach have of commercial AMF inoculants (Abbott, Robson, & Gazey, 1992) is shown variable outcomes (Farmer et al., 2007; Symanczik, Courty, important in order to properly assess cost/benefit analysis and to de- Boller, Wiemken, & Al-Yahya’ei, 2015). While Köhl, Lukasiewicz, and velop best practice protocols (Abbott & Lumley, 2015). Van der Heijden (2016) showed Rhizophagus irregularis establishment in a field soil microcosm, they did not measure persistence past 8 weeks. Earlier studies have shown persistence up to two (Pellegrino 6 | UNINTENDED CONSEQUENCES et al., 2012) and 3 years (Sykorova et al. 2015), but these studies also reported inconsistent establishment. Even if there is little evidence supporting the efficacy of AMF inocu- Long-term inoculation trials are clearly needed, but confirma- lants under field conditions, there is no evidence about the risk of in- tion of inoculant establishment will remain a challenge due to the oculation to ecosystems (Rodriguez & Sanders, 2015; Schwartz et al., inherent complexity of AMF genetic organization. AMF are known 2006). Inoculation with exotic AMF may have consequences for natu- to contain multiple haplotypes per isolate (Koch et al., 2004), which ral systems, particularly for fungal and plant communities. may lead to under-estimation of the inoculant if all haplotypes are not targeted by the approach. Within species, AMF strains can anas- 6.1 | AMF biodiversity tomose, which could lead to both an under-estimation of the inoculant fungi and a gradual dilution of effects (De La Providencia, De One of the few consistent lines of evidence is that inoculation can Souza, Fernández, Delmas, & Declerck, 2005). Furthermore, DNA change AMF communities, leading to partial (Koch et al., 2011; can persist in the environment in quiescent and dead cells (Levy- Symanczik et al., 2015) or complete replacement of resident fungi Booth et al., 2007) leading to the detection of inoculants that have (Jin, Germida, & Walley, 2013; Pellegrino et al., 2012; Symanczik et al., failed to establish. 2015). Not surprisingly, the degree to which an inoculant dominates HART et al. Functional Ecolo gy | 131

FIGURE 4 Decision tree highlighting the most relevant questions to ask when determining whether arbuscular mycorrhizal fungi (AMF) inoculation is necessary. The following conditions are likely to result in a negative impact AMF communities: soil removal, excessive tillage, extended fallow periods, continuous use of non-mycorrhizal crop(s), salinity, drought and some forms of contamination. The likelihood of passive re-establishment may increase with use of AMF host cover crops, and with dispersal from nearby sources of AMF. Broader goals such as crop yield, economic gains, C sequestration or the promotion of soil health should be considered prior to AMF inoculum usage depends on the context. For example, AMF with r-life-history strate- been shown to reduce plant biodiversity and/or prevent the establish- gies inoculated into communities of K-strategists could displace resi- ment of native plants (Emam, 2016; Koziol, Bever, & Hawkes, 2015; dent fungi (Abbott et al., 1983) particularly in disturbed (Antunes et al., Middleton et al., 2015; Torrez, Ceulemans, Mergeay, de Meester, & 2009) or stressful conditions (Symanczik et al., 2015). Abiotic factors Honnay, 2016). Interestingly, inoculant provenance has been shown can also influence inoculant dominance—inoculants may fail to estab- to be an important predictor of plant community response. For ex- lish effective levels of colonization in soils to which they are not well ample, native, rather than exotic inoculant AMF may help local plants adapted (Abbott et al., 1983). As with establishment, there is a lack of recover from herbivory (Middleton et al., 2015) or withstand plant in- data on long-term changes to AMF communities following inoculation. vasions (Burkle & Belote, 2015). In contrast, non-native AMF in com- As it stands, existing research indicates that the degree to which an mercial inoculants may lead to unwanted promotion of exotic over inoculant AMF alters resident communities may be difficult to predict. native plant species.

6.2 | Plant biodiversity 7 | THE ELEPHANT IN THE ROOM: ISN’T Inoculation studies that use plant diversity as a response variable EVERYTHING EVERYWHERE? overwhelmingly show that inoculation changes plant communities— which may be a desired outcome of inoculation (i.e. for landscape A frequent response to the potential risk of inoculation is that restoration), or an unintended consequence. AMF inoculants have AMF are globally distributed, so inoculation should be of little 132 | Functional Ecology HART et al. ecological consequence (Rodriguez & Sanders, 2015). But we know risk. For horticultural systems where plants are normally trans- little about the local and global distribution of AMF genotypes and planted into the field, natural inoculum from local soils would we are only beginning to understand the biogeography and evolu- be preferred to commercial inoculants. tionary biology of the Glomeromycota (Davison et al., 2015; Öpik, 2. Severely degraded soils: In some landscape restoration events, in- Metsis, Daniell, Zobel, & Moora, 2009; Powell & Bennett, 2015). In oculum potential is absent (i.e. pit mines, degraded agricultural particular, almost nothing is known about population-level dynam- soil). While practitioners could wait for natural AMF dispersal, in ics in AMF communities (Angelard, Colard, Niculita-Hirzel, Croll, & reality, successful restoration of AM systems requires AM plants Sanders, 2010; Koyama et al., 2017; Rodriguez & Sanders, 2015), to establish before non-AM plants. In this case, AMF inoculation but analysis of coding genes indicate that each isolate’s genome is may be necessary to help desired AM plants out-compete non- unique (Kamel, Keller-Pearson, Roux, & Ané, 2016). As such, inocu- mycorrhizal, ruderal plants. Again, research strongly supports lation with a novel genotype may lead to population-level changes. the use of local AMF inocula to negate risks from alien If these changes result in functional changes to AMF communities introductions. (i.e. changes to soil aggregation or to changes in nutrient uptake), then ecosystem functioning may also be affected (see Antunes & Given the lack of available data upon which to form best practises, Koyama, 2017). Such gaps in knowledge of AMF population and we suggest growers approach the use of AMF inocula conservatively, community ecology mean that a predictive framework for the fate and only when necessary (Figure 4). of inoculants is a long way off (see Box 1: What drives inoculant spread?) ACKNOWLEDGMENTS

M.M.H. was supported by a Killam Faculty Award, and a Gledden 8 | CONCLUSIONS Fellowship. Antreas Pogiatzis, Brittany Altwasser and Negin Kazemian collected data and created maps for Figure 1. Based on the available data, we conclude that the current practice of AMF inoculation is at best a gamble, and at worst an ecological threat: AUTHORS’ CONTRIBUTIONS Emphatically, most systems already contain functional AMF 1. M.M.H. conceived the idea and collected data. M.M.H., P.M.A., L.K.A. communities. Degraded systems may contain highly competitive (Cambridge, MA) led the writing of the manuscript. M.M.H., P.M.A., AMF that have proven tolerance to local conditions, which L.K.A. and V.B.C. contributed to writing the manuscript. V.B.C. devel- may be particularly resistant to competition from novel oped charts, figures and developed theoretical models. isolates. 2. There is as yet no evidence of “superior” fungal mutualists in the AMF symbiosis, and given the extreme context specificity of the DATA ACCESSIBILITY AM symbiosis, it is unlikely that such fungi exist, although there are All data are available as Supporting information in the online differences in fungal efficacy. version of the paper. 3. In the case of agriculture, the high nutrient levels in some cropping systems means that at least nutritionally, the AM symbiosis may not be limiting plant performance, and inoculants may fail to es- ORCID tablish at a functional level, or at all, because soil nutrients are too Miranda M. Hart http://orcid.org/0000-0002-2503-8326 high. 4. Inoculant users may not be able to limit their inoculants to their Lynette K. Abbott http://orcid.org/0000-0001-8586-7858 intended site, given that AMF propagules disperse through air, soil and water, and most AMF taxa can associate with most plants. Thus, if AMF inoculants establish, they have the potential to move. REFERENCES Abbott, L. K., Lumley, S. E. (2015). Mycorrhizal fungi as a potential indi- There remains much to learn about the biology and ecology of cator of soil health. In Z. M. Solaiman, L. K. Abbott & V. Varma (Eds.), Mycorrhizal fungi: Use in sustainable agriculture and land restoration (pp. AMF before we can adequately develop and control the use of AMF 17–31). Heidelberg, Germany: Springer. biofertlizers (Figure 3). Until then, we suggest that there are suitable Abbott, L. K., & Robson, A. D. (1982). The role of vesicular arbuscular my- conditions that warrant AMF inoculation: corrhizal fungi in agriculture and the selection of fungi for inoculation. Australian Journal of Agricultural Research, 33, 389–408. Abbott, L. K., & Robson, A. D. (1984). The effect of root density, inoculum 1. Horticulture: Closed systems, using artificial soil or hydroponics placement and infectivity of inoculum on the development of vesicular could benefit from AMF inoculum, since most horticultural crops arbuscular mycorrhizas. New Phytologist, 97, 285–299. are highly mycorrhizal. Additionally, as glasshouse agriculture is Abbott, L. K., Robson, A. D., & Gazey, C. (1992). Selection of inoculant a closed system, inoculants will not present an environment VAM fungi. In J. R. Norris, D. J. Read, & A. K. Varma (Eds.), Methods HART et al. Functional Ecolo gy | 133

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Research article Open Access Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus Daniel Croll and Ian R Sanders*

Address: Department of Ecology & Evolution, Biophore building, University of Lausanne, CH-1015 Lausanne, Switzerland Email: Daniel Croll - [email protected]; Ian R Sanders* - [email protected] * Corresponding author

Published: 15 January 2009 Received: 10 June 2008 Accepted: 15 January 2009 BMC Evolutionary Biology 2009, 9:13 doi:10.1186/1471-2148-9-13 This article is available from: http://www.biomedcentral.com/1471-2148/9/13 © 2009 Croll and Sanders; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory. Results: We addressed the question of recombination in the AMF Glomus intraradices by sequencing 11 polymorphic nuclear loci in 40 morphologically identical isolates from one field. Phylogenetic relationships among genotypes showed a reticulate network pattern providing a rationale to test for recombination. Five statistical tests predicted multiple recombinant regions in the genome of a core set of isolates. In contrast, five clonal lineages had fixed a large number of differences. Conclusion: Our data show that AMF from one field have undergone recombination but that clonal lineages coexist. This finding has important consequences for understanding AMF evolution, co-evolution of AMF and plants and highlights the potential for commercially introduced AMF inoculum recombining with existing local populations. Finally, our results reconcile seemingly contradictory studies on whether AMF are clonal or form recombining populations.

Background Molecular evidence for recombination was previously Arbuscular mycorrhizal fungi (AMF) form symbioses with found by analysis of the highly polymorphic BiP and the majority of plants and influence their species diversity rDNA sequences from Glomus intraradices and other AMF and productivity [1,2]. The symbiosis is thought to have [8]. Recombination was detected among sequence vari- existed ever since the colonization of land by plants [3]. ants present within single isolates. Due to the multige- Growth of AMF is thought to be entirely clonal by produc- nomic nature of AMF, recombination could have been ing asexual spores and no sexual reproductive structures restricted to nuclei co-existing in the same cytoplasm, have been observed. Based on this fact, and the suggested without recombination of DNA from genetically different 400 million years of morphological stasis [4], AMF were individuals [9,10]. In populations of two related species, suggested to be ancient asexuals [5]. But over the past dec- arbitrary genetic markers showed significant evidence for ade, the question whether AMF are asexuals or exhibit recombination [11]. Alternative explanations such as recombination has become a controversial issue [6,7]. recurrent mutations or sequences from contaminating

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microorganisms could also explain these results because and (2) use multiple sequence-based and population the AMF were not cultivated in clean culture prior to anal- genetics methods to test for recombination in AMF. ysis. In contrast, two studies suggested a strict clonal evo- Detecting recombination in AMF would be important lution in populations through analysis of multiple because it would further our understanding of a main fun- polymorphic loci from spores of field populations gal phylum, could have important consequences for [12,13]. In the first study, amplified fragment length pol- understanding the co-evolution of AMF with plants and ymorphism (AFLP) was scored for spores originating from could have far reaching consequences for the use of com- a single pot of a cultured AMF isolate. Genetic diversity mercial AMF inoculum. was found among spores but no evidence of recombination. The lack of replicate amplifications from single Results and Discussion spores makes it difficult to reach a definite conclusion. Multi-locus genotypes of G. intraradices population Furthermore, the material originated for each species from We used a set of 40 in vitro cultivated isolates of G. intra- a single pot culture and the fungi may not have had the radices originating from one population in Tänikon, Swit- opportunity to recombine with other genotypes from a zerland, to address the question of occurrence of field. In the second study, clonal reproduction in AMF was recombination in AMF. For reference, we included the iso- suggested by complete linkage of alleles at three loci late DAOM181602 originating from Québec, Canada. All among spores of field populations. Ideally, a much larger isolates were clonally subcultured to obtain sufficient number of polymorphic loci should be investigated to quantities of clean DNA [17]. Sequences of 11 polymor- draw conclusions about recombination. Using field-col- phic nuclear markers were used for a total alignment lected spores directly for genotyping would provide a length of 3037 bp. These markers were initially developed more representative sample of the actual genetic diversity to reveal sequence length polymorphisms and were iden- in an AMF population than using in vitro fungal cultures, tified using repeat-finding software. However, sequencing as factors such as host plants used during cultivation of the different alleles showed that the large majority of could bias the composition of successfully established iso- the detected polymorphism was found in regions flanking lates [14]. However, currently only the in vitro system pro- the repeat motifs. We found 75 polymorphic sites and 72 vides the required DNA quantities from fungi grown indel mutations (for details on polymorphism at each under sterile laboratory conditions necessary for reliable locus see additional file 1). The high degree of sequence genotyping at a large number of loci [15]. Croll et al. [14] polymorphism identified within the population corrobo- developed a set of 11 sequence-based markers to survey rates earlier data on polymorphism in coding regions genetic diversity and host plant preferences in a popula- [18,19] and random genetic markers [17]. The distinct tion of 40 G. intraradices isolates established in an in vitro combinations of alleles at the 11 loci identified 17 geno- cultivation system. The genotyping was based on length types in the population (labeled I–XI and XIII–XVIII). Iso- polymorphism at nuclear and mitochondrial loci and late DAOM181602 was a unique genotype not found in sequencing of all loci in representative isolates was used to the Swiss population (labeled XII). A sequence alignment confirm locus specificity of the genotyping method. How- of all 11 loci from the 18 genotypes is available as addi- ever, length polymorphism data alone are not suitable for tional files 2 and 3. Identifying organisms by multi-locus recombination tests as length homoplasy of distinct sequence typing was pioneered by Maiden et al. [20] for sequences could introduce a strong bias. Using sequence bacteria and successfully applied to fungi to e.g. infer information from all identified genotypes would allow a sources of human pathogen outbreaks [21]. variety of tests for recombination and could, therefore, be used to challenge the fundamental assumption of ancient Phylogenetic relationships among genotypes asexuality in AMF [16]. We first estimated the phylogenetic relationships among the genotypes to identify potentially recombining geno- In strict terms, members of a morphospecies of unknown types and clonal lineages. Then, a series of statistical tests reproductive mode found together in the same location were performed on sequences from all genotypes to test should not be called a population, as interbreeding of whether there is significant evidence of recombination in individuals is implied by the term population. For sim- the population. By applying a neighbour-network algo- plicity and in accordance with previous literature, we con- rithm [22] reticulate paths connecting the core genotypes tinue to use the term population to describe isolates of the could be observed (I–X, DAOM181602, XIII, XIV; Fig. 1). same AMF species found in one location. A reticulate pattern suggests that recombination among genotypes may have contributed to the evolution of the In this study, we use multi-locus sequence data of one G. genotypes, but multiple alternative mechanisms such as intraradices population established in an in vitro system to lack of phylogenetic resolution or homoplasy may also (1) resolve phylogenetic relationships among genotypes contribute to a reticulate pattern [23]. However, if recom- that would indicate recombination or clonal evolution, bination occurred within the population it is most likely

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DAOM 181602 (XII) III 99 XIII XIV II 91

100 VIII V 99 IX VI I IV VII X

100

XV 100 XVI

100

XVIII

XVII 0.001 PhylogeneticFigure 1 relationships among multi-locus genotypes of G. intraradices Phylogenetic relationships among multi-locus genotypes of G. intraradices. The neighbour-network using uncorrected p distance showed reticulate phylogenetic branching among a core set of genotypes. Substitutions and indel mutations were given equal weight in the analysis. Bootstrap support for branches above 90% is indicated in % of 1000 replicates. Roman numerals represent the different genotypes from one field (I–XI and XIII–XVIIII) and DAOM181602 (XII).

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among the core group of genotypes connected through a concatenation. In total, we used three different concatena- network (Fig. 1). The core genotypes weakly clustered into tion orders, two of which were random orders and the two subgroups, but multiple branches connect the two third separates loci with strong signals of recombination. subgroups (I, IV–X and II, III, XIII, XIV, DAOM181602 We found that five isolates from the population (II, III, XI, respectively; Fig. 1). Five genotypes (XI, XV–XVIII) were XIII and XIV) and the commercially cultivated isolate distinguished by a comparatively large number of muta- DAOM181602 showed recombination breakpoints tions from the set of core genotypes with very high boot- detected by two or more tests in all three concatenation strap support (100%; Fig. 1). Three more genotypes were orders. Furthermore, isolate XVIII showed recombination significantly differentiated from the core genotypes, but breakpoints by at least one test in all three concatenation branch lengths were considerably shorter (IX, orders. Isolate X did not show any recombination break- DAOM181602, XIV; Fig. 1). The reticulate pattern among points in any of the concatenation orders. The graphical the genotypes was conserved if all indel polymorphisms results showing significant recombination breakpoints are were excluded from the analysis (see additional file 4). shown in additional files 5, 6, 7. Detailed results with sig- This confirms that potential recurrent mutations, due to a nificance thresholds for each predicted recombination higher indel mutation rate observed in repeat-rich breakpoint of the concatenation order shown in addi- regions, do not qualitatively alter the results of the analy- tional file 5 can be found in additional file 8. The pre- sis. The reticulate pattern among the core genotypes was sented recombination breakpoint analysis is not suitable still observed when the strongly differentiated genotypes to distinguish between intragenic recombination or reas- (XI, XV–XVIII) were excluded, and this further supports sortment of loci as sequences of individual loci are too the evidence of recombination (see additional file 4). short to perform the tests on each locus separately.

Recombination detection in concatenated sequences Congruence analysis among loci To examine evidence of recombination, we used multiple Recombination creates conflicting phylogenies among sequence-based tests, because detection abilities can vary distant loci through reshuffling of DNA sequences among strongly among tests depending on the degree of poly- different genomes. To test this prediction, we used a fur- morphism and phylogenetic divergence of sequences ther, independent recombination test. The partition [24,25]. We performed six tests of recombination on con- homogeneity test creates artificial datasets by sampling catenated sequences of 11 loci. The concatenation of loci randomly among all observed sites of the genotypes and was necessary in order to overcome the limiting number then swapping sites among loci [33,34]. The length of of polymorphic sites within individual loci. Firstly, we maximum parsimony trees for all loci are calculated and used the Φw test that has been shown to reliably discrimi- summed. If recombination occurred among the genotypes nate between recurrent mutations and recombination in the population, the actual summed tree length should [26]. The test uses a sliding-window procedure to assess be shorter than the summed tree lengths based on the arti- phylogenetic compatibilities of nearby polymorphic sites ficially created datasets, because recombination should in the concatenated sequences. A total of 72 sites were have introduced incongruence among loci. The partition informative and significant evidence of recombination homogeneity test showed that the actual summed tree was found among the genotypes (p < 0.0001). We then length was 8 steps shorter than the shortest observed used two phylogenetic (Bootscanning and RDP) and three summed tree length in the artificially created dataset (p < nucleotide substitution based (Geneconv, MaxChi and 0.001, Fig. 2). Results were qualitatively similar if all indel Chimaera) tests [27-32]. These tests had already been polymorphisms were excluded (see additional file 9). applied to several fungal datasets in a critical analysis of Such conflicts in phylogenetic congruence among loci are statistical power of different recombination tests [24]. As most likely explained by recombination among the geno- sliding-window based tests are sensitive to the phyloge- types. The extent and frequency of recombination cannot, netic signals of neighbouring polymorphic sites, the con- however, be inferred from these results. Results from the catenation order of our loci is likely to affect the detection partition homogeneity test were shown to be sensitive to abilities of the different tests. A pair of concatenated loci bias in base composition and mutation rate across loci that would have been inherited clonally within the popu- [35,36], but as our study included individuals only from lation is not expected to show evidence of recombination. one population, we expect this influence to be negligible. However, for a pair of concatenated loci that show a reshuffling within the population, the tests are likely to Index of association among loci provide evidence for recombination (i. e. a significant The index of association measures the degree of linkage recombination breakpoint). As not all pairs of loci are among different loci. This test has been widely used to expected to show similar levels of clonal inheritance or detect linkage disequilibrium, following the initial appli- reshuffling, multiple concatenation orders are useful to cation to bacterial genotypes [37]. In our study, we calcu- control for a potential bias through the arbitrary nature of lated the index of association for the complete set of

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160

140

120

100

number of replicates 40 actual summed tree length = 154 20

0 152 154 156 158 160 162 164 166 168 170 172 174 176 178 180 182 summed tree lengths PartitionFigure 2 homogeneity test of 11 nuclear loci Partition homogeneity test of 11 nuclear loci. Partition homogeneity test showing the actual summed tree length by maximum parsimony of the dataset compared to summed tree lengths of 1000 artificially created datasets through re-sampling. The actual summed tree length is significantly shorter than the artificial datasets, indicating that incongruence exists among loci. Substitutions and indel mutations were given equal weight in the analysis. genotypes as well as for the two different subsets of geno- found no evidence for linkage disequilibrium in two types. According to the phylogenetic relationships among Glomus species based on ISSR loci. Our analysis showed the 18 genotypes (Fig. 1), including all 18 genotypes in that in a partially recombining AMF population, choosing the analysis of index of association would be unlikely to various subsets of genotypes depending on the evidence reveal recombination because of a number of lineages of clonality changes the outcome of linkage analyses, but that appear not to recombine with the core isolates. that groups of genotypes without significant linkage dise- Indeed, analysis using all 18 genotypes revealed strong quilibrium can indeed be detected. linkage disequilibrium (IA = 4.076, p = 0.0001), suggesting a population that deviates significantly from recom- Evidence for recombination in an AMF population binant. If the five genotypes showing the strongest evi- Taken together, the graphical and statistical tests of recom- dence for clonal evolution are excluded (see Fig. 1; bination strongly suggest that recombination occurred bootstrap value = 100%; genotypes XI, XV–XVIII among some of the genotypes in the field. The network excluded) the linkage disequilibrium is no longer signifi- analysis of the genotypes does not provide direct evidence cant (IA = 0.226, p = 0.087). No linkage disequilibrium for recombination, as other processes than recombination was detected either (IA = 0.167, p = 0.183) if all signifi- could lead to a reticulate pattern [22]. However, the anal- cantly separated genotypes are excluded (see Fig. 1; boot- ysis suggested that a core group of genotypes are the most strap value > 90%; genotypes IX, XI, XII, XIV–XVIII likely candidates to have undergone recombination. The excluded), indicating that this subset of genotypes is index of association analysis showed that, indeed, the potentially recombining. Two previous analyses of index strongest signal of recombination is among this core of association in AMF populations showed opposite group of genotypes. The analysis of phylogenetic congru- results from each other, although they studied different ence among all genotypes using the partition homogene- AMF species to those in the present study. Stukenbrock ity test strongly suggested the occurrence of and Rosendahl [13] found strong linkage among three recombination within the population. Furthermore, by loci in three Glomus species, suggesting a strict clonal evo- combining the results of the six sequence-based tests lution. Vandenkoornhuyse, Leyval and Bonnin [11] using a sliding-window analysis, five isolates of the popu-

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lation showed significant recombination breakpoints sup- isolates arise as a result of shifts in the relative frequency ported by two or more tests in all three tested of pre-existing nuclear variants. concatenations. Interestingly, the commercial inoculum DAOM181602 grouped into the core group of recombin- Clonal evolution and potential for cryptic speciation ing G. intraradices isolates, with evidence supported by Our study reveals that a five isolates of G. intraradices iso- multiple tests. Recurrent mutations or homoplasy are lates show strong evidence for recombination. However, unlikely to explain these results as excluding indel poly- we also identified five genotypes (XI, XV–XVIII) that were morphism and using robust methods do not qualitatively distinguished by a comparatively large number of inde- influence the results. A previous study of genetic diversity pendent mutational events, suggesting a clonal evolution in the same population used length polymorphism data in these lineages (Fig. 1). Statistical support for a clonal at the same loci as in this study to identify different iso- evolution of these genotypes was shown by very high lates [14]. The reticulate relationships among the different bootstrap values separating these five lineages from the genotypes suggested the occurrence of recombination as core group of genotypes (100%; Fig. 1). Several statistical pointed out by Young [16]. However, complete sequenc- tests identified putative recombination breakpoints in all ing of all loci in all genotypes and appropriate analyses these genotypes. However, these findings were not corrob- would be required to reach a clear conclusion [16]. orated by different concatenation orders (additional files Homoplasy in the dataset would artificially increase sig- 5, 6, 7). This suggests that recombination between these nals of recombination as a pair of genotypes showing alle- latter genotypes and other genotypes in the population is les of identical length but distinct sequence at a particular either absent or rare. However, a much higher sampling locus would be considered as potentially recombinant. effort in the field could yield genotypes that recombined The present study was based on sequences from all loci with those lineages. Very few morphological criteria are and all identified genotypes, therefore, allowing the con- known to distinguish isolates from the different clonal trol of size homoplasy. In future studies, data on genomic lineages, but the clonal lineages show clear differences in locations of the different loci could elucidate the fre- hyphal and spore densities produced in root organ cul- quency of recombination events and distinguish between tures [17]. Furthermore, genetic differences among a sub- inter- and intra-chromosomal recombination. set of the currently studied AMF population were shown to affect symbiotic functions with host plants [38]. Genetic heterogeneity in AMF Knowledge of the effects on host plants by different clonal An alternative hypothesis could potentially explain pat- lineages and recombining genotypes would be important terns of recombination in sequences, as in some AMF it to understand the potential effects of recombination on has been shown that isolates harbour genetically different AMF – host plant interactions. Recombination may have nuclei [9,10]. The previous use of linkage disequilibrium reduced genetic and phenotypic differentiation among analyses (i.e. index of association) on AMF spores using the core genotypes, while clonal evolution in the separate AFLP were shown to be misleading if only genetic varia- lineages may have led to the fixation of a large number of tion within isolates was considered [10]. The sensitivity of genetic differences. In order to consider genotypes of dif- fingerprinting techniques may artificially increase linkage ferent lineages as belonging to one biological species (i.e. disequilibrium and potentially mask recombination. forming an interbreeding population), all individuals Kuhn et al. [10] concluded that genetic heterogeneity should have the potential to undergo genetic exchange found among nuclei within isolates has most likely arisen among each other. Our data, however, suggests that sev- by accumulation of mutation, instead of recombination. eral lineages are independently evolving and may give rise To avoid the potentially confounding factor of within-iso- to cryptic speciation. late variation, we deliberately chose markers that did not reveal any significant within-isolate polymorphism (see Detection of recombination in supposed ancient asexuals Supporting Information in [14]). We have found other The detection of recombination in a population of AMF is markers showing multiple alleles within single spore iso- in strong contrast to the previous assumption of ancient lates. These were excluded from the analysis to rule out asexuality in the phylum of Glomeromycota. The main confounding within-isolate recombination but are the argument for asexuality was based on morphological sta- subject of further research. Thus, the genotypes included sis over 400 million years, suggested by fossil evidence, in our study describe the unique, or at least the over- and a lack of observed sexual structures [3,5]. Together whelmingly predominant, nuclear genotype present in with bdelloid rotifers and ostracods [5], AMF represent each isolate. The evidence for this is that only single alleles what Maynard Smith termed "evolutionary scandals" as were found at each locus within isolates using capillary accumulation of deleterious mutations and a slow rate of electrophoresis (see Supporting Information in [14]). This adaptation should strongly disadvantage asexual repro- argues against a potential explanation that is an alterna- duction in comparison to sexuality in the long term [39]. tive to recombination, namely that differences between While several theories tried to address this issue, recent

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evidence suggests that very few true asexuals might actu- this mutualism. Different AMF genotypes in the popula- ally exist [8]. A more likely scenario is that genomes of tion have different effects on plant growth [38]. Recombi- most organisms undergo at least sporadic recombination nation among different genotypes may, therefore, events to cope with the deleterious effects of long term produce AMF genotypes with novel effects on plant asexuality. Genomic signatures of recombination seem to growth. Furthermore, evidence for recombination in AMF be more pervasive than previously thought [8], highlight- has an impact on commercially harnessing the beneficial ing the fundamental role of sex in the evolution of traits of AMF for increasing crop production. The assump- genomes. tion of commercial AMF inoculum use is that an introduced AMF will not recombine with the local population. Sexual reproduction and vegetative incompatibility Our results highlight the potential that recombination Sexual reproductive cycles involving the fusion of geneti- could occur with the native AMF population and, by this, cally different hyphae (i.e. mating) are thought of being introduce new genes into the population. Finally, our the main mechanism in fungi allowing recombination findings reconcile previously published studies as recom- among different genomes [40]. But the lack of laboratory bination and clonality were both detected in AMF. evidence of sexual structures involved in mating proved to be inconclusive about the occurrence of recombination in Methods the species. Over the past decade, molecular evidence Origin of fungal isolates strongly suggested the occurrence of recombination in a A total of 40 G. intraradices isolates using in this study number of fungal species that were presumed to strictly originated from an agricultural field site in Tänikon, reproduce clonally. The human pathogen Coccidioides Switzerland [51]. The same set of isolates was used for immitis, the pathogenic Aspergillus flavus and A. fumigatus, population genetic analyses using AFLP, simple as well as the ant cultivated fungi in the fungus-growing sequence repeat and mitochondrial markers [14,17]. ant symbiosis were all shown to undergo at least sporadic Species identity of all isolates was verified by Croll et al. recombination events or even to possess functional genes [14] by sequencing the internal transcribed spacer region required for meiosis [41-44]. In the case of Cryptococcus and subsequent comparison with deposited sequences neoformans, where a known sexual cycle exists, population of the same species. The G. intraradices isolate genetic structures were found to be almost completely DAOM181602 originated from a field site at Pont clonal. Nevertheless, reproduction between individuals of Rouge, Quebec, Canada (Biosystematics Research Cen- the same mating type was shown to allow recombination tre, Ottawa, Canada). G. intraradices is widely used as a to occur [45]. In a related species, a similar mechanism commercial AMF inoculum for agricultural applications was shown to be at the origin of a major human pathogen (Premier Tech Inc., Canada) and is the first AMF isolate outbreak [46]. to be entirely sequenced [52]. It is a haploid fungus with a compact genome of 15 Mb and is the only AMF for In AMF, vegetative incompatibility was shown to occur which ploidy has been measured [53]. Ploidy could be when mycelia from different locations come into contact different for other AMF species [54]. [47,48]. Such mechanisms, in combination with a lack of a known sexual cycle, were thought to exclude the possi- In vitro cultivation and DNA extraction bility of recombination between genetically different iso- In vitro cultures of each isolate were established from sin- lates [49]. Our study suggests that these mechanisms need gle spores with Ri T-DNA-transformed carrot root on to be further researched within AMF populations as they standard M growth medium [55]. Five to ten two-com- are the most likely mechanisms allowing the mixing of partment plates were inoculated by clonal subculturing to nuclei and subsequent recombination among different allow the proliferation of the fungus in one compartment AMF in the soil. that is kept root-free, while remaining connected to the roots in the other compartment [17,56]. Root-free fungal Conclusion compartments of all plates were pooled per single spore Evidence for recombination in AMF has further implica- line for extraction of hyphae and spores. Freshly isolated tions than simply to remove these fungi from the list of hyphae and spores of each isolate were separately dried putative ancient asexuals. Whether AMF are truly asexual overnight at 48°C and ground into a fine powder using a or form recombining populations is critical to under- Retsch MM300 mixer mill (Retsch, GmbH). The DNA was standing AMF co-evolution with plants. Stability in mutu- extracted using a modified version of the Cenis method alism can be favoured by asexuality of one partner, but for fungal DNA extraction with an additional step of 1:1 this does not allow the symbiont to rapidly adapt to dilution with a solution of 24:1 of chloroform isoamyl changes in the host [50]. Our study, therefore, has conse- alcohol before the final precipitation, to remove remain- quences for understanding the evolutionary potential of ing impurities [57].

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PCR amplification of polymorphic loci Phylogenetic analysis and tests for recombination We applied tests of recombination on 18 different G. The neighbour-net algorithm implemented in SplitsTree intraradices genotypes that were identified by Croll et al. 4.8 was used to construct a network based on the uncor- [14] on the basis of variation at 11 nuclear loci. In the rected p distance using concatenated sequences of all loci Swiss population, 17 genotypes were identified. The iso- [22]. The robustness of the branching pattern was assessed late DAOM181602 showed a unique genotype. Ten loci with 1000 bootstrap replicates. Analyses were performed were shown to contain short regions of repetitive DNA, in two ways: (1) coding indel and repeat polymorphisms indel polymorphism and SNPs in the flanking regions of with Gapcoder using 1/0 to code for presence or absence the repeats (see Supplementary Material in Croll et al. of gaps in the alignment [58] and (2) ignoring all indel [14]). In addition, one nuclear gene intron was used [14]. polymorphisms (see additional file 4). Ignoring gaps The function of the gene is currently unknown. Each reduces the risk of including polymorphisms that may be marker revealed only one allele per single spore isolate due to recurrent mutations occurring in short repeat [14]. We identified other markers with multiple alleles per motifs, confounding potential signatures of recombina- isolate but chose not to include these in the analysis to tion. Nevertheless, the majority of indel mutations are not avoid confounding potential intra-isolate recombination in repetitive stretches of DNA and are, therefore, unlikely with among isolate recombination (data unpublished). to be recurrent mutations. Croll et al. [14] identified alleles at each locus based on length differences and sequenced each allele that was seen Following Posada [24] and Posada and Crandall [30], we to be different due to length polymorphism. For this applied several recombination tests to evaluate the robust- study, we extended the sequencing, by sequencing all 11 ness of our results. To test for evidence of recombination loci in all the 18 genotypes identified by Croll et al. [14] based on phylogenetic compatibilities of nearby polymor- so that any additional polymorphisms that were not phic sites along concatenated sequences, the Φw test based on length differences could be detected and also to implemented in Splitstree 4.8 was used [26,59]. The test check whether alleles with the same length in different was run with the default settings of a window size of 100 isolates were also the same sequence. PCR amplifications and k = 2. Five additional recombination tests were run in were performed according to Croll et al. [14]. PCR prod- order to predict putative recombinant regions in the con- ucts were purified using the MinElute PCR purification kit catenated sequences: (1) Geneconv [29,32] was per- (Qiagen, Inc.). Purified and quantified PCR products were formed scanning sequence triplets and treating indel directly cycle sequenced with BigDye Terminator v1.1 blocks as single polymorphisms. (2) MaxChi [28] was (Applied Biosystems, Inc.) following the supplier's used to scan all possible sequence triplets with 30 variable instructions. Cycle sequence products were purified by sites per window, alignments gaps (indels) were not con- ethanol precipitation. An ABI PRISM™ 3100 Genetic Ana- sidered as this could generate false positives [27]. (3) Gen- lyzer was used for automated sequencing. All sequence eral recombination detection (RDP test implemented in profiles were visually checked using 4 peaks software (A. RDP software; [27]) was performed with a window size of Griekspoor and T. Groothuis, http://mekentosj.com/ 10 and without specifying a reference sequence. (4) Boot- 4peaks). Sequences of all loci were aligned using the Clus- scanning [31] was used with a window size of 100 and a talW algorithm implemented CLCbio Free Workbench step size of 20, standard distances were used for calcula- 4.0 software http://www.clcbio.com and alignments were tions and p values were binomial. (5) Chimaera [30] is a checked manually. Independent PCR and re-sequencing modification of the MaxChi test and was run with 30 var- of alleles was performed to check for potential genotyping iable sites per window. All five tests were run with RDP errors for all loci and in several isolates. No sequence var- v2.08 [27], setting the significance threshold to p = 0.05. iation was found in these tests. Identical labels (roman Multiple comparison corrections were performed by a numerals I–XVIII) were used to name the genotypes as in Bonferroni correction [27]. the previous study [14]. The sequences at all 11 loci of the 18 genotypes were deposited: locus Bg32 [GenBank: Partition homogeneity test EU534209–26]; Bg42 [GenBank: EU534227–44]; Bg62 The partition homogeneity test [33,34] implemented in [GenBank: EU534245–62]; Bg196 [GenBank: PAUP 4.0b10 [60] was used to estimate the degree of EU534263–80]; Bg235 [GenBank: EU534281–98]; Bg273 incongruence among loci created by recombination. To [GenBank: EU534299–316]; Bg276 [GenBank: compare the degree of incongruence, 1000 datasets were EU534316–34]; Bg303 [GenBank: EU534335–52]; Bg348 artificially created by re-sampling all sites without replace- [GenBank: EU534353–70]; Bg355 [GenBank: ment, regardless of the loci boundaries. The summed tree EU534371–88]; nuclear intron [GenBank: EU534389– length of the maximum parsimony trees of each locus 406]. A sequence alignment of all genotypes with concate- found for the original dataset was compared to the nated loci as used for the data analyses is available in addi- summed tree lengths of the maximum parsimony trees of tional files 2 and 3. each locus for the artificially created dataset. Firstly, the

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analysis was performed giving equal weight to substitutions and indel polymorphisms, giving a total of 77 parsi- Additional file 5 mony-informative sites. An additional 70 variable sites Summary of five recombination tests based on the concatenated were parsimony-uninformative. Secondly, the analysis sequences of 11 nuclear loci. Loci are concatenated arbitrarily according to the locus labelling. For individual tests, recombinant regions can over- was performed considering only substitutions in the lap as all significant recombinant regions were kept in the analysis. Sig- sequences. This provided 32 parsimony-informative char- nificance was based on p < 0.05, corrected for multiple comparisons. For acters and 43 parsimony-uninformative characters (for exact p values for all putative recombinant regions see additional file 2. results see additional file 9). The total alignment length is 3037 bp. Click here for file Index of association [http://www.biomedcentral.com/content/supplementary/1471- 2148-9-13-S5.pdf] The index of association (IA) measures the degree of linkage equilibrium among a set of genotypes. IA = VO/VE -1, Additional file 6 where VO is the observed variance of the number of loci Summary of five recombination tests based on the concatenated being different within all pairs of genotypes and VE is the sequences of 11 nuclear loci. Loci Bg62, Bg196 and Bg235 showing variance of the number of loci being different within all strong signals of recombination in additional file 5 are not consecutive. pairs of genotypes expected under complete linkage equi- Click here for file librium. Therefore, I = 0 if genotypes recombine freely. [http://www.biomedcentral.com/content/supplementary/1471- A 2148-9-13-S6.pdf] The IA and the significance threshold of a deviation from linkage equilibrium (with 10'000 randomizations) were Additional file 7 calculated with the program MultiLocus v1.3b developed Summary of five recombination tests based on the concatenated by P.-M. Agapow and A. Burt [61]. sequences of 11 nuclear loci. Loci are concatenated in a 2nd arbitrary order compared to additional file 5. Authors' contributions Click here for file DC conceived the study, performed the molecular and sta- [http://www.biomedcentral.com/content/supplementary/1471- tistical analyses and drafted the manuscript. IRS partici- 2148-9-13-S7.pdf] pated in the design of the study and helped to draft the Additional file 8 manuscript. Details of the recombination breakpoint analysis with the Recombina- tion Detection Program on concatenated sequences of the 11 loci Additional material among 17 genotypes of one field of Glomus intraradices and the isolate DAOM181602. The concatenation order corresponds to the one presented in additional file 5. Additional file 1 Click here for file Polymorphism found in sequences of 11 loci among 17 genotypes of [http://www.biomedcentral.com/content/supplementary/1471- Glomus intraradices from one field and the isolate DAOM181602. 2148-9-13-S8.pdf] Click here for file [http://www.biomedcentral.com/content/supplementary/1471- Additional file 9 2148-9-13-S1.pdf] Partition homogeneity test of 11 nuclear loci with all indels being removed. Additional file 2 Click here for file Sequence alignment of 11 concatenated loci of 18 genotypes of G. [http://www.biomedcentral.com/content/supplementary/1471- intradices. Different shades indicate different loci. 2148-9-13-S9.pdf] Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-9-13-S2.pdf] Acknowledgements Additional file 3 We thank Rui Rodrigues Martins Candeias for help with in vitro cultivation Sequence alignment of 11 concatenated loci of 18 genotypes of G. and DNA extraction. The Swiss National Science Foundation is acknowl- intraradices in fasta sequence format. edged for financial support with a grant to I.R.S (number 3100AO-105790/ Click here for file 1). Laurent Keller, Nicolas Salamin and Alexander M. Koch provided helpful [http://www.biomedcentral.com/content/supplementary/1471- comments on the manuscript. 2148-9-13-S3.txt] References Additional file 4 1. Smith SE, Read DJ: Mycorrhizal Symbiosis. San Diego: Academic Phylogenetic relationships among genotypes in a population of G. Press; 1997. intraradices based on a Neighbour-network using uncorrected p dis- 2. Heijden MGA Van der, Klironomos JN, Ursic M, Moutoglis P, Streit- tance. wolf-Engel R, Boller T, Wiemken A, Sanders IR: Mycorrhizal fungal Click here for file diversity determines plant biodiversity, ecosystem variability and productivity. Nature 1998, 396(6706):69-72. [http://www.biomedcentral.com/content/supplementary/1471- 3. Redecker D, Kodner R, Graham LE: Glomalean fungi from the 2148-9-13-S4.pdf] Ordovician. Science 2000, 289(5486):1920-1921.

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The Ecology of Arbuscular Mycorrhizal Fungi

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Critical Reviews in Plant Sciences Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bpts20 The Ecology of Arbuscular Mycorrhizal Fungi A. Willis a , B. F. Rodrigues b & P. J. C. Harris a a Centre for Agroecology and Food Security, Coventry University, Coventry, United Kingdom b Botany Department, Goa University, Goa, India Version of record first published: 30 Nov 2012.

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The Ecology of Arbuscular Mycorrhizal Fungi

A. Willis,1 B. F. Rodrigues,2 andP.J.C.Harris1 1Centre for Agroecology and Food Security, Coventry University, Coventry, United Kingdom 2Botany Department, Goa University, Goa, India

Table of Contents

I. INTRODUCTION ...... 2

II. TAXONOMY ...... 2

III. EVOLUTION ...... 3

IV. HYPHAL NETWORK ...... 3

V. PHENOLOGY ...... 4

VI. SOIL AGGREGATION ...... 6

VII. PROPAGULE DISSEMINATION ...... 6

VIII. TRANSPORT ...... 7

IX. PHOSPHORUS ...... 7

X. CARBON ...... 7

XI. NITROGEN ...... 8

XII. MICRONUTRIENTS ...... 8 Downloaded by [A. Willis] at 20:31 04 December 2012

XIII. COLONIZATION ...... 8

XIV. MICROBIAL INTERACTIONS ...... 8

XV. INVASIVE PLANTS ...... 9

XVI. PHYTOREMEDIATION ...... 10

XVII. EFFECTS UPON AM FUNGI ...... 11 A. Disturbance ...... 11 B. Agrochemicals ...... 11 C. Grazing ...... 12

Address correspondence to A. Willis, Centre for Agroecology and Food Security, Coventry University, Priory Street, Coventry CV1 5FB, UK. E-mail: [email protected]

1 2 A. WILLIS ET AL.

D. Parasitism: The Case for Trichoderma harzianum (Ascomycota) ...... 12 E. Global Climate Change (GCC) ...... 12 F. AM - AM Competition ...... 13 G. A Little P Goes a Long Way ...... 14

XVIII. CONCLUSIONS ...... 14

REFERENCES ...... 14

also evidence of ammonium- and nitrate-nitrogen contribution Arbuscular mycorrhiza is a mutually beneficial biological as- derived from organic matter (OM) (Leigh et al., 2008). Sec- sociation between species in the fungal phylum Glomeromycota ondary roles of AM fungi include reduction of root invasion by and higher plants roots. The symbiosis is thought to have afforded microbial soil-borne plant pathogens (Newsham et al., 1995), green plants the opportunity to invade dry land ca 450 Ma ago and reduction in plant uptake of phytotoxic heavy metals (Gohre¨ the vast majority of extant terrestrial plants retain this association. Arbuscular mycorrhizal (AM) fungi perform various ecological and Paszkowski, 2006), improved host plant water balance in functions in exchange for host photosynthetic carbon that almost periods of ample water and drought (Auge,´ 2001) and soil par- always contribute to the fitness of hosts from an individual to com- ticle aggregation through the cohesive action of a Glomalean munity level. Recent AM fungal research, increasingly delving into water-stable glycoprotein (Rillig and Mummey, 2006). Further the ‘Black Box’, suggests that species in this phylum may play a effects of AM reported include reduction in insect herbivory key facilitative role in below-ground micro- and meso-organism community dynamics, even more perhaps, that of a bioengineer. by induced plant response (Bennett et al., 2009) and variation The ubiquitous nature of the symbiosis in extant flora and the fact in that response relative to nitrogen (N) uptake (Gange et al., that variations from the AM symbiosis are recent events suggest 2005), increase in insect pollination (Gange and Smith, 2005) that Glomeromycota and plant roots coevolved. This review con- and percentage increase in Fı generation seed germination (Sri- siders aspects of AM fungal ecology emphasizing past and present vastava and Mukerji, 1995). AM are also reported to increase importance of the phylum in niche to global ecosystem function. Nutrient exchange, evolution, taxonomy, phenology, below-ground the density of insect herbivore parasites in trophic food webs microbial interaction, propagule dissemination, invasive plants in- (Hempel et al., 2009; Hoffmann et al., 2010). All of these func- teractions, the potential role in phytoremediation and some of the tions are performed in exchange for host plant carbon (C). There factors affecting AM fungal biology are discussed. We conclude is evidence to suggest AM fungi may play a significant role in that it is essential to include AM association in any study of higher soil N and C cycles (Govindarajulu et al., 2005; Jones et al., plants in natural environments in order to provide an holistic understanding of ecosystems. 2009) and make considerable contribution to terrestrial ecosystem C sinks (Wright and Upadhyaya, 1998). In addition to the Keywords arbuscular mycorrhiza, Glomeromycota, ecological com- above functions, AM fungi can also influence, perhaps even or- plexity, plant community driver, soil community facilita- ganize and structure, plant community patterns (van der Heijden tor, keystone mutualist et al., 2008) and soil microbiota community populations (Rillig Downloaded by [A. Willis] at 20:31 04 December 2012 et al., 2006; Toljander et al., 2007).

II. TAXONOMY I. INTRODUCTION Taxonomy of AM fungi describes 214 species in four orders, Arbuscular mycorrhizal (AM) fungi are ubiquitous root sym- 13 families and 19 genera, in the class Glomeromycetes of bionts of more than 90% of vascular plants and over 80% of all the phylum Glomeromycota (Muthukumar et al., 2009), sur- extant terrestrial plants (Wang and Qui, 2006). Arbuscular myc- prisingly few considering their having been extant for 450 Ma orrhiza has been found in the Rhynie Chert, an Early Devonian (Pirozynski and Malloch, 1975) or more (Redecker et al., 2000). sedimentary deposit exposed near the village of Rhynie, Ab- The phylum is represented worldwide in almost all major terres- erdeenshire in Scotland, which formed 410 Ma ago (Remy et al., trial biomes (Treseder and Cross, 2006), in bryophytes (mosses), 1994). AM fungi are considered to have been indispensable in hepatics, pteridophytes, gymnosperms and angiosperms. Al- green plant colonization of terrestial habitats (Helgason and Fit- most all tropical plants are typically AM mycotrophic (Janos, ter, 2005). Their primary function is thought to be contribution to 1987), a phenomenon that may be related to rapid litter plant nutrition, particularly phosphorus (P) (Bolan, 1991), often decomposition and consequent high ecosystem C turnover a limiting resource, and micronutrients (Clark and Zeto, 2000), (Cornelissen et al., 2001). Only the plant families Chenopo- especially in nutrient depleted hostile environments. There is diaceae, Polygonaceae, Juncaceae, Cruciferae (Brassicaceae), ARBUSCULAR MYCORRHIZAL FUNGI 3

Caryophyllaceae and Proteaecae do not consistently host AM be reasonable to assume they were early in AM fungal evolu- fungi (Smith and Read, 2008). The fungal species are consid- tionary history. All genera are globally represented and dispersal ered non-host plant specific in their associations (Giovanetti of hosts and fungal symbionts was perhaps enhanced by con- and Hepper, 1985), although there appear to be clear cases tinental drift through the formation and break-up of the super- of preference (Vandenkoornhuyse et al., 2002; Croll et al., continents Pangea and Gondwanaland. The fossil record does 2008). not substantiate this, however, there being no reliable represen- Traditionally the species have been identified by morpholog- tation of taxa other than ‘Glomus-like’. Nicolson (1975) de- ical characteristics of spores and sporocarps, spore suspensors scribed AM fungi in a Late-Carboniferous gymnosperm species and subtending hyphae, but increasingly sophisticated molec- resembling those in extant species. Stubblefield et al. (1987) re- ular methods are now also used. PCR analysis has become a ported fossilized arbuscules structurally similar to modern-day regular feature of species identification since the isolation and AM fungi in Triassic strata in Antarctica. There are a number amplification of an AM fungi small subunit rRNA (SSU-rRNA) of reports of AM fungi in Quaternary deposits. 18S gene sequence by Simon et al. (1992). Universal primers Even if the fossil record is extended, it is unlikely to show have been developed that enable AM identification to species how and when AM fungal species diversified. Consideration of level in both soil and root samples with negligible error, and geological history, however, suggests that since origination AM gene-code reference libraries are readily available on CD-ROM fungi have survived five major extinction events, after the last of and the internet. The subsequent development of a quantita- which previously slowly spreading flowering plants rapidly di- tive real-time PCR analysis technique (Alkan et al., 1994) has versified. Genotypic change in evolving host plants on a global enabled expansion in research into spatial, temporal and func- recovery scale after each extinction event may have driven fun- tional symbio-biological activities of AM in planta and in the gal symbiont speciation beyond background levels. mycorrhizosphere (Robinson-Boyer et al., 2009; Konig¨ et al., Thus, 80% of all extant terrestrial plants, including species 2010). of ancient lineage, bryophytes (mosses) (Zhang and Guo, 2007) though there are contrary reports, liverworts (Duckett et al., 2006), hornworts (Schußler,¨ 2000), quillworts (Radhika and Rodrigues, 2007), club mosses (Winther and Friedman, 2008), III. EVOLUTION selaginellas (Strullu-Derrien and Strullu, 2007), horsetail ferns The earliest fossil evidences of AM fungi are isolated spores (Dhillion, 1993) and cycads (Muthukumar and Udaiyan, 2002) from the Ordivician of Wisconsin dated at 460 Ma ago, and in and more than 90% of extant vascular plants including ancient Early Devonian Rhynie Chert where features similar to extant conifers and Old World angiosperms are arbuscular mycorrhizic. Glomus spp. hyphal and arbuscular structures were found in There are degrees of dependence upon the symbiosis on the part the protostelic roots of Rhynia and Asteroxylon, early vascular of the higher plant species ranging from obligate to facultative plant species dated at ca 410 Ma. As these features appear to to non-mycorrhizal mycotrophy within and between species. be evolutionarily advanced, arbuscular mycorrhiza may have Emerging ecto-, ericoid- and orchid-mycorrhizas, and non-AM by then been evolving for a considerable period. It is likely evolutionary developments, such as fine roots with root hairs, that the symbiosis developed with primitive freshwater-aquatic cluster roots and non-mycotrophic plants, are all relatively re- phototrophic gametophytes (Embryophyta) long before the Or- cent events occurring from the Mid- to Late-Cretaceous through dovician invasion of dry land and the development of myc- the Tertiary (95–2 Ma) period (Brundrett, 2002), so it is likely Downloaded by [A. Willis] at 20:31 04 December 2012 orrhizal rhizoidal bryophytes and hepatics. This is supported that almost all plants up to this time were to some extent AM my- by evidence from Wang et al. (2010) describing three genes cotrophic. AM symbiosis has thus had a considerable effect on required for AM formation isolated from almost all ancient global plant community ecosystems during extreme oscillations plant lineages, indicating AM presence in the common ances- in environmental conditions for at least 365 Ma. tor of land plants. A further indication of ancient ancestry is the AM symbiosis between the cyanobacterial species Nos- toc punctiforme and the only living representative of the ancient Geosyphonaceae, Geosyphon pyriformis. Redecker and IV. HYPHAL NETWORK Raab (2006) suggest Geosyphon (Archaeosporales) is closely There are two distinct types of AM fungi, characterized by in- related to basal Archaeospora, branching-off earlier than Para- traradical hyphal modifications: (i) the Paris-type where hyphal glomus (Paraglomales) in the phylogeny of the phylum. Sub- development is exclusively intracellular, forming coils in host sequent origins of Glomerales, the largest order representa- plant cortical cells, and (ii) the Arum-type, where intraradical tive in the phylum, and Diversisporales are indicated as be- hyphal development is mostly intercellular and forms arbuscules ing monophyletic (however, see Walker and Schußler¨ (2002) in root cortical cells (Figure 1). These are the characteristic tree- http://invam.caf.wvu.edu/index.html [23.11.10], who suggest like structures from which AM fungi derive their name. Between convergent evolution may have occurred in Diversisporales). these ends of the spectrum are a number of gradations, described When origins and divergences occurred is unknown but it might as intermediate types (Dickson, 2004). 4 A. WILLIS ET AL.

FIG. 1. Sketch of inter-radical morphological features of AM fungi. (a) = appressoria; (ar) = arbuscules of intercellular Arum-type AM; (ar-c) = arbusculate-coil and (c) = coil of intracellular Paris-type AM; (s) = inter-radical spore; (v) = vesicles.

Smith and Smith (1997) in their literature survey found 41 is no clear positive relationship between soil mycelial biomass angiosperm families to have only Paris-type, 30 only Arum- and the quantities of nutrient transferred to hosts (Smith et al., type and 21 families with examples of both. Gerdeman (1965) 2000), nor to other AM fungi functional attributes. reported a Paris-type mycorrhiza in tulip tree but Arum-type There is evidence of nutrient transfer, P (Whittingham and in maize from the same fungal isolate. Coils and arbuscules Read, 1982; Wilson et al., 2006), N (Cheng and Baumgartner, were observed by Kubota et al. (2005) in the same root sys- 2004; Motosugi and Terashima, 2006) and water (Allen, 2007), tems of cucumber and tomato. The structures are thought to be between inter- and intra-specific host plant above-ground tis- nutrient exchange sites, at least from fungus to host, including sues via AM fungal hyphae, a facilitative function that may pro- phosphate and ammonia/ammonium transporter systems via an foundly affect plant relationships (Selosse et al., 2006). Sim- H+-ATPase pathway across a specialized membrane formed in ilarly translocated C remains within the fungal structures in cortex cells (Kobae and Hata, 2010). The sites are connected recipient host roots (Bago et al., 2000). Interestingly Simard to a mycelial web in the extraradical sphere by inter- and intra- et al. (1997) reported transport of labelled-C from above-ground cellular hyphae. plant tissue to above-ground plant tissue via common ectomy- Downloaded by [A. Willis] at 20:31 04 December 2012 Taxa other than species in the families Gigasporaceae, Para- corrhizal hyphal connections. Observations on isolates of three glomaceae and Archaeosporaceae produce inter- and/or intra- AM Glomus species made by Giovannetti et al. (1999) sug- cellular lipid-rich vesicles. These possibly act as temporary gest that fungal genetic material may be commonly transferred storage organs for the fungi, sometimes converting to spore- through extraradical hyphal anastomosis in some taxa. Signif- like thick-walled structures, and vesicles may be important in icantly, geographically separated isolates of the same species the efficacy of root fragments as propagules. The extraradical appeared not to anastomose. Intraspecific anastomosis has not mycelial networks of many species have been shown to maintain been observed. There is no evidence of plant or fungal genetic viability if soils remain undisturbed, even after hot-dry or cold material being transferred from and to host plants yet this is a conditions, although the inoculum potential generally decreases potential pathway and there are many examples of symbiosis with time. These networks can be extensive and Miller et al. having transferred genes to the chromosomes of the host cell (1995) reported a maximum of 111 m cm−3 soil in tallgrass (Emiliani et al., 2009). prairie. This represents ca 0.002% of the soil volume based on an average mycelium diameter of 5 µm (Abbott and Robson, 1985). Hyphal lengths of <1–26 m g−1 have been reported in a V. PHENOLOGY variety of soils (Sylvia, 1992), and species differ in the degree of Arbuscular mycorrhizal fungal growth and development is soil volume occupied (Abbott and Robson, 1985) and distance dynamic and rapid. The asymbiotic stage, which is the only grown from host plant roots (Munkvold et al., 2004). There stage in the phenology of the organism where there is evidence ARBUSCULAR MYCORRHIZAL FUNGI 5

of limited saprophytic ability (Azcon-Aguilar´ et al., 1999), displays the lowest metabolic rate. The germ-tube of a spore may grow up to 20–30 mm, but if a host root is not contacted within as much as 15–20 days it may cease growth and become septated after metabolites are withdrawn. The spore may produce another germ-tube or remain quiescent until germination triggered by root proximity occurs, a strategy that conserves spore energy resource. At the pre-symbiotic stage, root exudate encourages germ-tube growth toward the root (Sbrana and Gio- vannetti, 2005) and triggers fan-shaped germ-tube branching (Tamasloukht et al., 2003) stimulating multiple entry points into the root. It may be that the spore is not the principal infective unit in thriving habitats, however, mycorrhizal root fragments and active hyphal networks being more effective (Smith and Read, 2008). Appressoria are formed at pre-determined intracellular points of contact with the root that have responded to fungal-derived signals to form prepenetration apparatuses (PPA) (Genre et al., 2005) through which penetration into the cortex occurs. Arbuscules are dichotomously highly-branched hyphae in entire surface contact with accommodating plant cell plasma membrane where the periarbuscular membrane (PAM), the site of nutrient exchange, is formed. The structure thus has a vastly FIG. 2. General schematic of AM fungi life-cycle. Root fragments may propagate from inter-radical spores, vesicles or hyphae. Species temporal and spatial increased exchange area. Arbuscules develop within 1–6 days variations occur in response to differing and changing inter- and extra-radical of penetration into cortex cells (Harley and Smith, 1983). When environmental influences. fully developed they occupy, for example, 35% and 36% of the cells in wheat and oats, respectively (Alexander et al., 1988). After 4–15 days, the arbuscules degenerate and the host cell and Read, 2008). This implies that photoassimilates are ex- returns to its original state (Harley and Smith, 1983). Kobae changed via the PAM. Mosse and Hepper (1975) reported ex- and Hata (2010) recorded only 2–3 days of active phosphate traradical hyphal growth immediately upon colonization of root transport in transgenic rice host roots before arbuscule degener- in monoxenic culture, before the formation of arbuscules, which ation. Further arbuscules develop as intercellular hyphae spread suggests intraradical hyphae may also be exchange sites for host through the root and continue to penetrate receptive cortical plant C. Runner hyphae extend alongside and around the root cells. Re-entry into previously occupied cells has been ob- apically and distally, re-inoculating. Nutrient absorbing feeder served. Total percentage of root length occupied by arbuscules hyphae extend out into the matrix bridging rhizosphere nutrient varies with fungal species (Fitter, 1985), season (Bohrer et al., depletion zones. They branch dichotomously up to eight times 2004), edaphic factors (Clark, 1996; Posada et al., 2007), soil narrowing in diameter from ca 20 to 2 µm (Friese and Allen, hydrology (Schreiner et al., 2007) and soil temperature (Smith 1991) enabling uptake of nutrient resource from the smallest Downloaded by [A. Willis] at 20:31 04 December 2012 and Read, 2008). The extent of root colonization also varies with soil crumbs inaccessible to root hairs. The extensive mycelial soil biota interactions (Dauber et al., 2008) and with host plant web formed interconnects the root systems of plants of different species (Klironomos, 2003), host phenological stages (Pongrac species, genera and families directly or through hyphal anas- et al., 2007) and C allocation (Muthukumar and Udaiyan, 2000). tomosis (Giovannetti et al., 2006). The life cycle is completed There is comparatively little similarly detailed description with the production of spores (Figure 2). of Paris-type coils in the literature. van Aarle et al. (2005), Glomeromycota spores are multi-nucleate, heterokaryotic investigating G. intraradices in two different plant species, (Hijri and Sanders, 2005), and formed asexually (Pawlowska, reported levels of metabolic activity in Paris-type colonization 2005). These are unusual features that raise intriguing questions in one plant similar to that in Arum-type in the other. Kobae and about speciation (Redecker and Raab, 2006) and adaptation to Hata (2010), by a novel fluorescence technique in dual-type change. Microscopical determination of variation in wall mor- Gigaspora rosea colonization of rice, found no evidence of a phology, shape, color, size and reaction to staining compounds specific Pi transporter that was upregulated on membrane sur- such as Melzer’s reagent and Sudan Black B, has been the rounding the fine branches of arbuscules and smaller arbuscular traditional method of classification, now enhanced by molecu- structures in arbusculate coils, on membrane surrounding coils, lar methods. The spores are multi-walled and large, generally the hyphae of arbusculate coils, or arbuscule trunks. visible to the naked eye, and range from <40 µm in the small- The extraradical mycelia branch and extend into the rhizo- est species to >800 µminGi. gigantea. All spores contain sphere and beyond when arbuscules have been formed (Smith hundreds-to-thousands of nuclei that may not be genetically 6 A. WILLIS ET AL.

identical, and lipid globules from which developing germ-tubes VII. PROPAGULE DISSEMINATION in the pre-symbiotic stage draw the majority of their required Evidence clearly indicates that germinating spores and active energy. Spores are formed singly, in clusters or in sporocarps AM mycelial webs colonize plant roots within the immediate in the soil, and in some species within root tissue. Numbers are mycorrhizospere and that each web overlaps and interacts with − typically 1-<50 g 1 soil (Sjoberg¨ et al., 2004). Rarely are more all others in the vicinity, spatially becoming a ‘global’ network. than 20–25 taxa reported in ﬁeld studies. Density and distri- A strategy for dissemination of viable AM fungal propagules bution vary both spatially and temporally within and between over distance is less clear. There are reports of spores being vec- species, with soil types and with host plants species diversity. tored by insects and small mammals, and possibly by water and An AM fungal species spatial sporulation-patch framework was wind over long distances. The latter would certainly have been observed by the authors during research in sand dunes on the an advantage in the spread of early seed-bearing plants with west coast of India where fresh spores of Scutellospora gregaria long distance dispersal mechanisms. However, no viable spores were the most abundant in a 0.25 m2 Zoysia matrella patch at the to date have been recovered from an apparatus devised to cap- end of the monsoon season in one year, with few found in the ture wind-borne AM fungal propagules at a 0.5-m height over an same period of the following year when a Gigaspora species 18-month period at Goa University (Willis and Rodrigues, un- was dominant (Willis and Rodrigues, unpublished). Viability published data). Wind-borne AM hyphal fragments are reported longevity may be enhanced by a dormancy mechanism that is to be non-viable. This is supported by the consistant failure of little understood, but is assumed to assure longer-term survival. hyphal fragments collected from the apparatus to colonize host Experimental evidence strongly suggests all AM fungal roots in trap culture. The question of wind-borne viable propag- species are adapted to locality, with isolates from one geograph- ule dissemination was re-visited after observations made on ical location often exhibiting poorer functional performance in fresh iron-ore mine spoil mounds where there was spontaneous other areas (Pellegrino et al., 2010; Antunes et al., 2011). As sporadic growth of herbaceous AM fungal colonized plants. a consequence, consideration should be given to the source of The plants may have been inoculated by raindrop splash from spores used as inoculum in sustainable agricultural practice and a nearby, lower, line of colonized plants developed from dump- in ecological restoration. truck wheel deposits (India west coast monsoon rains can be very heavy at times) or perhaps by inoculated root-invaded leaf litter carried in from adjacent restoration plantings. Aristizabal´ VI. SOIL AGGREGATION et al. (2004) reported AM hyphal and vesicle colonization of There is considerable evidence that the Glomalean hydropho- leaf litter in Colombian montane sites, and litter containing AM bic glycoprotein glomalin is involved in soil-crumb aggregation colonized roots used as inoculum in trap culture at Goa Univer- (Wright and Upadhyaya, 1998). Large quantities of this recalci- sity soon heavily colonized host roots (Willis and Rodrigues, trant material are deposited from active extraradical hyphae and unpublished data). released during degeneration, and act as a mucilaginous glue The sporadic spontaneous mycorrhizal plant growth pattern contribution to binding soil matrix. Soil micro-aggregates are observed in the mine spoil indicates an epi-central plant commu- bonded more tightly than macro-aggregates (>250 µm) (Smith nity development strategy in primal, hostile and nutrient-poor and Read, 2008), perhaps suggesting greater glomalin deposi- soil environments, but only on a limited scale initially. The tion from the larger surface areas of small diameter hyphae. niche in which a viable spore or root fragment is deposited by The colonization of soil by microbiota and subsequent incor-

Downloaded by [A. Willis] at 20:31 04 December 2012 chance necessarily needs to be an environment which facilitates poration of detrital organic materials develops and maintains a AM development. This should include actively growing plant structurally water-stable living soil. roots, an N source and associative saprotrophic microbial pop- Glomalin C fraction can range from 9–22%, residence time in ulations which contribute to labile nutrient pools. Where AM soil from 6–42 years, and may represent >5% of total soil C (Ril- development occurs, the surviving plants will contribute to soil lig et al., 2001a). AM fungi thus have immense influence upon nutrient status through rhizodeposition, litter-fall and root death soil C cycles. Rillig et al. (2005) conducted long-term meso- leading to a gradual below-ground increase in hyphal networks. cosm research with single AM fungal species isolates. They This accretion strategy may be the principal form of AM fungal reported differences in aggregation that they attributed to the dissemination. influences of different associative microbiotic communities. In Supporting evidence for the hypothesis is limited but there a mixed AM community, this may indicate a highly complex are data which, by inference, may substantiate the notion. A biotic heterogeneity in mycorrhizosphere soils. Interestingly re- database constructed from reports of worldwide SSU-rRNA searchers consistently report higher values of available P in the AM fungi sequences by Opik¨ et al. (2010) indicated limited smallest aggregates, in Ultisols (Thao et al., 2008) and in highly distribution ranges of most taxa but geographically wide weathered laterite soils (Wang et al., 2001), regions in the matrix ranges where host taxonomic range is also wide, distribution unavailable to plant roots and root hairs but available to hyphae, patterns which may well have arisen from an accretion process. an exploitation advantageous to AM fungi and consequently Similarly, AM inoculated mixed-species herbaceous and host plant fitness. woody ‘primer’ plants, spaced as fertile islands in managed ARBUSCULAR MYCORRHIZAL FUNGI 7

re-instatement programmes in impoverished landscapes, spread others requirements effecting a balance or equilibrium. Environ- and interact with each other through rhizosphere accretion. mental change, which may occur at the niche level, in localized Growth of hyphal networks varies with AM fungal species disturbance, and in global ecosystem oscillations, necessarily but can be rapid and extensive, found mostly in the top ca 20 cm moves the relationship away from this equilibrium. Each part- volume of soil. Allen et al. (2003) estimated, in Glomus spp. ner reacts compensatively to maintain fitness, yet still within a studied, every 1 mm of new root growth equated to formation framework of synergism. of 122 cm of hyphae. Roots can expand at the rate of several cm day−1. Any successive AM mycotrophic plant propagules IX. PHOSPHORUS arriving within a developing or established mycorrhizosphere All species of AM fungi contribute inorganic P (P ) to their have immediate access to the benefits of the symbiosis, expo- i hosts, particularly in a P limited environment. The extent of nentially increasing the productivity of the network season by P supply varies depending on the colonizing fungal symbiont season, year by year. The arrival of novel AM fungal and host species (Smith et al., 2010). There is evidence of hydrolysation species would contribute to the development (or disturbance) of of organic P (P ) by AM extraradical hyphae at the hyphal tip complex multi-species above- and below-ground communities. o (Koide and Kabir, 2000), but there are very limited quantities If accretion is the primary method of AM dissemination it of AM fungal derived phosphatase in soils adjacent to hyphae may help in explaining within-species spore tolerance or adap- (Joner et al., 2000). Other soil microorganisms, bacterial and tation to environment. Often it is found spores of the same AM fungal, with abundant hydrolysis capacity, release copious quan- fungal species collected from geographically different locations tities of P into the labile pool that AM fungi trans-membrane have differing influences on plant fitness. As the fungi encounter i transport into extraradical ‘feeder’ hyphae (Karandashov and variation in heterogeneity of local environments during expan- Bucher, 2005), convert to polyphosphate long-chain and gran- sion, isolates, those species that generally perform better than ular fractions (Solaiman et al., 1999), and transport to the in- reference species in ‘home’ soils, may commonly develop. traradical exchange sites by cytoplasmic streaming (Cox et al., 1980). Cox et al. (1980) quantitatively assessed mean P flux in extraradical hyphae at 2.7 × 10−8 mol P cm−2 s−1. VIII. TRANSPORT Temporal aspects of plant nutrient demand require atten- Both intra- and extra-radical hyphae are generally non- tion when considering the supply role of AM fungi. Harper septate. Formation of septa has been noted in hyphal-branching (1977), for example, suggested “there is evidence in cereals structures immediately prior to root contact (Giovannetti et al., (which exemplify annual strategies) that 90% of total nitrogen 1993), in the process of repair (de la Providencia et al., 2005), and phosphorus content of the mature plants has been absorbed at arbuscule degeneration, on separation of functional and de- before the plant has achieved 25% of its final dry weight” and generating intraradical hyphae (Kinden and Brown, 1975), and Radhika (2008) found two of the commonly occurring herbs during germ-tube retraction. Septa in sub-tending hyphae are studied had maximum root and shoot P concentrations during an important feature in morphological spore identification in flowering stage and a third species during the vegetative stage. some species. The transport of materials within hyphae is bidirectional (Smith and Gianinazzi-Pearson, 1988). Soil derived nutrients are transported to the host while the C assimilates X. CARBON from the host utilized by the fungus are distributed throughout Carbon supplied from the host to the fungal symbiont is Downloaded by [A. Willis] at 20:31 04 December 2012 the mycelial web and hyphosphere. Nutritionally the symbio- derived from plant sugars and is thought to be transported by sis might be described as a loop, a bottom-up or top-down (or passive efflux. The intraradical hyphae and/or arbuscules take both) feedback system whereby each partner benefits. The fun- up hexose, a substantial amount of which is used in lipid, tre- gus delivers nutrients from the available pool in excess of its halose and glycogen synthesis before translocation to extrarad- own minimal maintenance requirements and thus enhances the ical mycelia (Bago et al., 2000; Bago et al., 2003). Up to 20% fitness of the host. Similarly the host provides C in excess of of total photosynthate, always of recent assimilate partitioned its own minimal maintenance requirements and thus enhances to roots, may be supplied to the fungus. Movement of lipid the fitness of the obligate symbiont. Where the fungal partner bodies from intraradical to extraradical hyphae has been im- delivers nutrients to the host, physiological changes such as aged by real-time immunofluorescence technique (Bago et al., increase in transpiration and photosynthetic rates occur (Allen 2002a). Other work has also shown movement in the opposite et al., 1981), enabling the host to return excess C to the fungus. direction (Bago et al., 2002b). Much of this C is utilized in fun- Where, however, the host has sufficient nutrients from other gal maintenance and growth and there is evidence that the AM sources such as agricultural P fertilizers, C supply to the fungus mycelial web releases C into the mycorrhizosphere (Toljander is reduced, and colonization and diversity is decreased (Johnson et al., 2007), just as roots exude into the rhizosphere soil matrix, and Pfleger, 1992). Optimally, in both natural and sustainable influencing biota populations (Jones et al., 2009). Estimates of agriculture systems, the partners would benefit most where each soil C derived from AM fungal extraradical hyphae range from is able to simultaneously and constantly deliver nutrient to the 50 to 900 kg ha−1 (Zhu and Miller, 2003). Rillig (2004) reported 8 A. WILLIS ET AL.

1.45 Mg C ha−1 complexed in glomalin in the top 10 cm of soil Daucus carota with G. intraradices was halved when supply of in a lowland tropical forest. the nutrient was increased above growth limiting levels.

XIII. COLONIZATION XI. NITROGEN Several species of a number of genera of AM fungi may Comparatively recent investigation has shown AM fungi to colonize a host root simultaneously (Bever et al., 2001). Al- play an important role in the transport of N from OM and leaf though these species are, by definition, competitive with each litter (Leigh et al., 2008) to host plants. Unlike with P, AM other (Wilson, 1984; Smith and Read, 2008) there may be com- fungi do not enhance the acquisition of N when present at low plementarity, as suggested by Jansa et al. (2008). Variation in levels in soil (Reynolds et al., 2005) but can make a significant colonization strategy is displayed at the taxonomic level (Hart contribution to plant N requirement (Hodge and Fitter, 2010), and Reader, 2002) with each species possibly contributing dif- particularly in dry soils where mobility to the direct pathway fering functions to the symbiosis to varying degrees. Arbuscular via roots is restricted (Tobar et al., 1994). The hyphal pathway mycorrhizal communities within a host can change significantly converts inorganic N taken up from the labile pool into amino over time. The species that dominate in saplings have been acids, and translocates it principally as arginine from extrarad- shown to become minor species as the host plant develops and ical to intraradical hyphae (Govindarajulu et al., 2005). Here grows while formerly rare or previously undetected species can the N is converted to inorganic N compounds before passing to become dominant (Husband et al., 2002). This may represent the host. A recent report by Guether et al. (2011) describes the r- and K-strategies, a colonization/persistence trade-off (Hart characterization of an organic N transporter in Lotus japonicus et al., 2001) on the part of some of the fungal species but it roots induced by mycorrhization that may be involved in active may also suggest that the hosts’ functional demands of the sym- transfer of organic N compounds, principally energy rich amino biont may change with time. For example, P and N demand may acids, to the plant. be of primary importance during early stages of plant develop- An increased P was consistently associated with an increase ment, particularly in nutrient deficient and hostile environments, in N accumulation in mycorrhizal Vicia faba under low P condi- whereas difficulties in transpiration may be of primary concern tions (Jia et al., 2004). As with all other aspects of mycorrhizal in mature canopy trees. ecology, however, N relations are complex. Jurkiewicz et al. Symbiotic events occurring in a woody host species may (2010) found that G. intraradices performed well in the colo- be quite different from those in an adjacent herbaceous species. nization of Arnica montana in high N soil, but no mycorrhization Even among mixed herbaceous species occurring events may be occurred when N was low. Blanke et al. (2005), on the other dissimilar even though their roots occupy common soil volume hand, reported strong colonization by AM fungi in P-polluted (Vandenkoornhuyse et al., 2003). Events in two adjacent plants N-deficient soils and reduced mycorrhization in comparable P of the same species may be quite different, particularly where level soils with higher N concentrations. AM fungal extraradical an AM fungal species might also exhibit functional variability hyphae are thought to contribute indirectly to leguminous plant (Smith et al., 2004). If, as has been recently suggested, the pri- N status but only in reduced P conditions, supplying essential mary driver of local adaptation of AM fungi is edaphic resource P and micronutrients to nitrogen-fixing organisms (Smith and availability (Johnson et al., 2010), it is feasible that ecological Read, 2008). activity in the symbiosis is as heterogenous as the between- and

Downloaded by [A. Willis] at 20:31 04 December 2012 within-soils matrices occupied. Perhaps plasticity derived from the inherent heterokaryotic nature of multiple nuclei is a strat- XII. MICRONUTRIENTS egy enabling isolate-variable AM fungal ﬁtness in almost any The symbiosis also contributes micronutrients to the host habitat, or niche. plant. Suzuki et al. (2001), using a multitracer technique, detected the uptake and transport to hosts of sodium (Na), zinc (Zn), selenium (Se), rubidium (Rb) and strontium (Sr) by AM XIV. MICROBIAL INTERACTIONS fungal hyphae. Burkert¨ and Robson (1994) observed Zn uptake Fungi are thought to have originated some 760 Ma −1.06 Ga to varying degrees in three fungal species and Caris et al. (1998) ago and Ascomycota 500–650 Ma ago (Lucking¨ et al., 2009). reported uptake of iron (Fe) in sorghum (but not in peanut) by Glomeromycota arose from non-septate Zygomycotina, an ear- G. mosseae. Marschner and Dell (2006) found that AM symbio- lier sister clade to septate Asco- and Basidio- mycotina, and are sis could account for up to 60% of plant copper (Cu) and 10% ancestrally symbiotic with all extant plant clades. This suggests potassium (K) requirements in experimental chambers. Clark the phylum evolved along with phototrophic charophytes, green and Zeto (2000) found that K, calcium (Ca) and magnesium algae which include the closest living relative of embryophyte (Mg) uptake was enhanced by mycorrhization in acidic soils plants, in primeval fresh-water and muds for perhaps as long as and Li et al. (2006) reported an increase in plant shoot Cu in 300–540 Ma before the advent of the invasion of dry land. It is calcareous soil, relative to P uptake. Allen and Shachar-Hill not possible to deduce whether AM fungi had, and subsequently (2009) found that uptake of sulphur (S) in monoxenic culture of lost, saprotrophic ability, or evolved to take advantage of a ARBUSCULAR MYCORRHIZAL FUNGI 9

symbiotic C source from the outset. Neither is it clear which of the labile pool. It is possible there is synergistic activity, di- the many functions now known to be attributable to AM fungi rectly or indirectly, between AM fungi and dark septate endo- were then imparted toward phototroph fitness. phytes (DSE), root inhabiting species of Ascomycetes and Ba- At the origins of AM fungi there would have already ex- sidiomycetes characterized by distinctive coil, knot and hyphal isted considerable archaean and bacterial species diversity. Blue- structures in cortex cells which may themselves be symbiotic green bacterial stromatolites have been dated as far back as 3.45 with higher plants. Observations made by Willis and Rodrigues Ga and fossilized biofilm along with spherical and rod-shaped indicate a clear reduction in root hair density, a universal phe- structures are present in 3.3–3.5 Ga old cherts in the South nomenon in the AM symbiosis, when roots are colonized by African Onverwacht Group (Westall et al., 2001). Interactions DSE, even in the absence of AM fungi (unpublished). with these organisms must have been as much a part of successful evolution of Glomeromycota as adaptation to the symbiotic habit. XV. INVASIVE PLANTS Much of the data on AM fungal interactions with other mi- Invasive plants, those which threaten native biological diver- crobial soil organisms, particularly rhizobacteria, has been ob- sity, employ a variety of tactics to gain a foothold and thrive in tained from trap-culture or in vitro studies. Care should be taken new territories (Sakai et al., 2001), including interactions with in interpreting these data as they may not fully represent the AM fungi. Marler et al. (1999), in a greenhouse experiment, complexities of the multi-trophic environment of soils. Never- concluded the invasive European mycorrhizal forb spotted theless insight into many significant saprotrophic and synergistic knapweed (Centaurea maculosa) was “strongly enhanced” associations has come to light. Increased supply of P to plants in interspecific competition with the North American native via AM hyphae has been observed in a large number of exper- grass species Festuca idahoensis only when mycorrhizae were iments where species of phosphate-solubilizing bacteria have present. Intraspecific competition was weak. They reported, been cultured along with the fungus. Similarly, detrital decom- in interspecific competition without AM fungi, F. idahoensis posers and nitrogen-fixing bacteria have been shown to enhance biomass gains up to 171% higher than with AM fungi, and plant N nutrition via AM mycelial uptake. It might be concluded C. maculosa plants 66% larger in interspecific competition that the fungus is ‘simply in the right place at the right time’ with AM fungi as against without. Walling and Zabinski but investigation by a number of researchers strongly suggests (2004), in a split-pot experiment with single plants on one side at least some species of AM fungi promote selection and pro- and P amendments in bare soils on the other, found greater liferation of specific bacteria in the mycorrhizosphere and of extraradical hyphal mass in association with C. maculosa those attached to extra-radical hyphae. This may result in an than with F. idahoensis, which suggests an increasing mineral enhanced AM fungal mycelium-available nutrient status in the nutrient exploitation potential. This is supported by previous labile pool. Mycorrhiza helper bacteria (MHB), species that as- work from Zabinski et al. (2002) that showed P concentrations sist mycorrhization and functioning of the symbiosis including in C. maculosa grown in a 28-µm membrane split-pots ex- plant root pathogen protection, have been investigated. Species cluding fine roots but not AM hyphae, adjacent to native grass of plant growth promoting rhizobacteria (PGPR) encourage the species, were increased. Mummey and Rillig (2006) described mycelial growth of AM fungi and, to a lesser extent, diazotrophic a significant (ave. 24%) reduction in extraradical hyphal lengths endophytic plant growth promoters in both fungal organs and in C. maculosa dominated sites as against native grass species plant roots, which may also be involved in interaction activity. dominated sites in the field. They didn’t differentiate between Downloaded by [A. Willis] at 20:31 04 December 2012 Interestingly there is clear indication that while there is little living and dead hyphae and so could draw no conclusions about or no specificity between host plants and AM fungi there may AM fungal function but suggested the resulting decrease in be specificity between bacterial species and AM fungal species. glomalin deposition and its effect on soil structure may be a It is thought specific bacterial diversity is a response to sub- factor accounting for greater erosion losses that occur in C. tle changes in rhizo-deposited photosynthates by the fungi. For maculosa dominated communities (Lacey et al., 1989). a comprehensive review of the plant growth promoting inter- Further research by Callaway et al. (2003) investigated the actions between bacteria and AM fungi, see Artursson et al. effect of invasive C. melitensis on two co-occuring native grass (2006). species Avena barbata and Nassella pulchra where application Soil fungi other than Glomeromycota have antagonistic, syn- of the fungicide benomyl had reduced AM fungal abundance. ergistic and neutral interactions with AM fungal species. Frac- They reported C. melitensis biomass reduced by >50% in fungi- chia et al. (1998) found one species inhibiting AM fungal spore cide untreated soils as against treated when grown alone, little germination and germ-tube growth while all others observed change in biomass when grown with A. barbata whether fungi- enhanced growth and root colonization of G. mosseae.TheAM cide treated or not and an almost 5x increase when grown with N. fungal species had reciprocal effects upon the associated sapro- pulchra where the resident AM fungal population had remained phytic fungi. Further complexity was observed with variation fungicide untreated. in both host species and light levels. These saprotrophs also Callaway et al. (2008) attributed inhibition of native contribute AM-available and hence plant-available nutrients to AM fungi in N. American forest soils by the European 10 A. WILLIS ET AL.

non-mycorrhizal invader garlic mustard (Alliaria petiolata, egy emphasizes the importance of plant species selection at Brassicaceae) to a ‘novel weapons’ effect, specific biochemicals the very beginnings of managed indigenous plant community released into the rhizosphere to which AM fungi in home restoration. soils are immune. The inhibition of native AM fungal species corresponded with severe adverse effects on the native plants community. In prior A. petiolata work in a series of experiments, XVI. PHYTOREMEDIATION Stinson et al. (2006) described an indirect effect of allelopathic Plants display a number of methods of averting possible suppression of AM spore germination and colonization and its stress due to heavy metals (HM) in contaminated soils. For adverse effect on mycorrhizal-dependant canopy tree seedling an overview at the molecular level see Hall (2002). There is recruitment. Burke (2008), also investigating A. petiolata evidence that some species of Glomeromycota in certain cir- interactions with AM fungi, on this occasion in three N. cumstances have a beneficial influence on plants growing in American native woodland herbaceous species, found the soils contaminated with HM by impeding uptake. Other species invasive selectively affected inhibition of a significant indicator encourage a higher rate of uptake that, if it occurs in tolerant Acaulospora AM fungal species in one of the native plants, plants, may aid detoxication of the contaminated soils by the while the AM fungal communities in the other two plants were process of phytoremediation. unaffected. In a different experimental approach, Anderson Gonzalez-Ch´ avez´ et al. (2004) found that glomalin in hyphae et al. (2010) removed A. petiolata from an invaded site, finding and in soils had sequestered the “potentially toxic elements increase in mycorrhizal inoculum potential and cover of native (PTEs)” Cu, cadmium (Cd) and lead (Pb). Interestingly they plants species within a two-year period. found no differences in sequestration by Cu tolerant over non- Barto et al. (2010) conducted a novel laboratory experiment tolerant G. mosseae isolates in vivo. Hyphal wall sequestration introducing A. petiolata root and leaf extracts at lower, more has also been reported (Joner et al., 2000) with HM binding realistic levels than in previous investigations, to another N. to chitin. Chen et al. (2005) described a correlation between American native woodland-floor plant, Impatiens pallida.They Pb sequestration and increased vesicle numbers. Ultra et al. defined four different growth stages: germinating seeds in the (2007) reported a reduction in arsenic (As) toxicity symptoms absence of AM fungi; seedlings growing where AM fungi were in Glomus inoculated sunflower plants. They also found that excluded (pre-symbiosis phase); germinating seeds and subse- As was converted to organic forms in the mycorrhizosphere, quent development with AM fungi (symbiosis formation phase); suggesting an active reducing mechanism by the AM fungus. and plants which had been colonized by AM fungi for four weeks A further mechanism is described by Gohre¨ and Paszkowski (symbiosis growth phase). Their results showed a ca 50% reduc- (2006) where a number of AM fungus produced chelators within tion in seed germination in the absence of AM fungi; reduction the cytosol bind the metals which are then pumped out by HM in root length and inhibition of root-foraging structure develop- transporters. Tullio et al. (2003) found that AM isolates from ment in the pre-symbiosis phase; growth rates of height, root Cd polluted sites impeded translocation of Cd to a greater extent and rhizosphere areas unaffected by the extracts in the symbiosis and colonized barley roots to a greater extent than spores from formation phase; and growth rate, root and shoot dry masses and non-polluted sites. root to shoot ratio unaffected by A. petiolata extracts in the sym- Experimental work in pots reported by Weissenhorn et al. biosis growth phase. They concluded that a pre-established AM (1995) clearly showed the complexity of AM fungal involve- fungal symbiosis in I. pallida ameliorates allelopathic effects of ment in HM translocation in plants. They conducted a multifac- Downloaded by [A. Willis] at 20:31 04 December 2012 A. petiolata and restoration of invaded areas more successful if torial study of the translocation of Zn, manganese (Mn), Cu, Cd colonized plants are used rather than the traditional method of and Pb from soils surrounding a disused smelting plant in maize sowing seed. plants at different light intensities. They found lower levels of The examples cited are few (for a comprehensive review HM (Cd, Cu, Zn, Mn) translocation where spores were from the see Pringle et al., 2009) but clearly they show AM fungi can contaminated soils and higher levels of inoculation compared contribute to the success (and failure) of invasive plants estab- to imported spores at increased light levels, again suggesting lishment. The evidence suggests in undisturbed terrestrial envi- fungal adaptation or at the least, spore tolerance. In the highest ronments many invasive plants are only able to gain purchase in levels of light-enhanced inoculation and plant biomass, how- new habitats by altering native AM fungal population density ever, AM isolates from the contaminated soils increased Cu and diversity, with consequential effects on native plant com- and Zn root-to-shoot translocation. Shen et al. (2006) noted an munities. It also lends considerable support to the proposal that interactive effect of Zn and Cd in mycorrhizal plant growth. Glomeromycota mediate in manipulation of plant community Although there appears to be spore tolerance to HM, abun- structure. dance itself may be adversely affected. Ortega-Larrocea (2001) Invasive plants regularly proliferate in disturbed areas, found a significant reduction in Glomus spores in clay-deep ver- rapidly modifying soil biotrophic communities, to the exclu- tisols where the HM chromium (Cr), nickel (Ni), Cu, Zn and Pb sion, perhaps, of endemic primary colonizer species. Ensuing had accumulated in agricultural fields after 90 as compared to plant community succession is biased at the outset. This strat- 5 years of wastewater irrigation. ARBUSCULAR MYCORRHIZAL FUNGI 11

Clearly there is no commonality in the role of AM fungi in ei- Millner, 1999; Oehl et al., 2003). Continuous monoculture of ther HM sequestration or phytoremediation. There is, however, maize and to a much less extent crop rotation also showed re- obvious potential. In certain circumstances and with careful duction in diversity. Soils left fallow or under sustainable or management it may be possible to produce edible fodder/food organic agricultural systems showed significantly greater diver- crops in HM contaminated soils. There may certainly be scope sity (Oehl et al., 2003; 2004). The greatest diversity was found for enhanced soil phytoextraction programmes utilizing metal in semi-natural grasslands. Hijri et al. (2006) reported similar hyperaccumulators, plants tolerant of very high levels of HM results from molecular studies of the same sites as the Oehl et al. in tissue (Kramer,¨ 2010), inoculated with appropriate HM tol- (2003) research. This evidence may suggest AM species diver- erant AM fungal strains. The particularly high occurrence of sity and abundance could be an ‘indicator’ of the fertility status hyperaccumulation in the Brassica family is excluded from en- of sustainable and organic agricultural soils. Where disturbance hancement by AM inoculation but Trotta et al. (2006) found is severe, such as removal and stockpiling of topsoil, AM fungal that As hyperaccumulation in the roots of Chinese brake fern propagule viability is considerably reduced after just three to (Pteris vittata) was reduced and translocation to above-ground four years of storage (Gould and Liberta, 1981). tissue increased when inoculated with G. mosseae and Buendia- Gonzalez´ et al. (2010) reported hyperaccumulation of Cr and B. Agrochemicals Cd in Prosopis laevigata (Fabaceae), which is known to be ar- Applications of agricultural chemical fertilizers, fungicides buscular mycorrhizal. Sarma (2011) states >500 plant species and pesticides have been shown to have both negative and posi- from 101 families have been documented as metal hyperaccu- tive effects upon AM fungal population characteristics. Increase mulators. in levels of plant available P by fertilizer application almost always promotes a negative feedback, reducing diversity and XVII. EFFECTS UPON AM FUNGI abundance in AM fungal community. Residual levels of P were found to inhibit AM fungal root colonization even after con- A. Disturbance version to organic systems (Hijri et al., 2006). An interesting Physical disturbance of the top 20–30 cm of soil drastically exception is described by Johnson (1984) where G. intraradices affects inoculum potential in the short term (Jasper et al., 1989) colonization of Citrus aurantium was unaffected after 26 weeks as the AM fungal mycelial web is fragmented. With the pos- following weekly application of P, and more root cortex sporu- sible exception of fragile, low nutrient-available systems such lation at the highest of concentrations. as sand dunes and arid regions, the recovery period appears to The literature on the effects of N fertilizer applications gives be rapid. Hyphal repair, inoculum potential of both hyphal and a less clear picture. Early work at Rothamsted Experimental root fragments, and re-connection of viable hyphal fragments Station, Harpenden, UK suggested increasing levels of nitrates via anastomosis may accelerate recovery. Arbuscular mycorrizal generally reduced AM colonization levels in lettuce (Owusu- species diversity recovery response to restoration after fire was Bennoah and Mosse, 1979), and in onion (Wang and Hayman, rapid where mycotrophic understorey herbaceous plants were 1982), the latter also contrastingly reporting no effect of am- re-planted (Korb et al., 2003). Winter freezing had little impact monium nitrate on AM colonization in Trifolium repens.Ba˚ath˚ on the inoculum potential of AM fungal hyphae (Addy et al., and Spokes (1989), investigating various combinations of P and 1997). Diversity also recovered rapidly where herbaceous plants N application effects upon the growth response of G. caledo- re-established on newly created islands after flooding in a Euro- nium in Allium schoenoprasum (chives), reported highest root Downloaded by [A. Willis] at 20:31 04 December 2012 pean alpine river (Harner et al., 2011). Recovery of diversity in colonization when N and P were at intermediate levels, no ef- agricultural soils in flood plains and deltas, for example annual fect on the addition of N at low soil P levels nor at high P and floods in the Gangetic Plains of India, is also rapid, possibly low N, and lowest levels of colonization at high P and high N, due to the relatively large diversity of spores at 50–75 cm in where ammonium-N had a greater effect than nitrate-N. Douds agricultural soils (Oehl et al., 2005). Alluvial deposits are also and Schenck (1990) found sporulation in Gi. margarita coloniz- a possible source of viable inoculum replenishment. Miller and ing Paspalum notatum (Poaceae) reduced, severely so in higher Bever (1999), in their study of AM fungal species variation in a concentrations, by ammonium nitrate, yet root colonization was wetland grass species along a dry-to-wet gradient, found certain little affected. Johnson et al. (2003) concluded that granular am- species in the drier regions only. There were no species found monium nitrate effects on AM in grasslands in different biomes only in the wet regions. There are flooding effects reported that were N:P site-dependant, a general reduction in C allocation to reduce AM colonization and spore numbers in rice paddy but AM structures in ample ambient P conditions, and an increase sufficient inoculum survives to colonize (19–33%) subsequent in P-deficient soils. non-flooded crops (Wangiyana et al., 2006). Fungicides captan and benomyl, the latter often applied in In the long term, for example in agricultural practices of comparative field studies on AM fungi, were observed to de- continuous tillage disturbance and in increasing land use inten- crease metabolic activity in AM fungal tissue as soon as three sity, AM fungal spore numbers and morphologically assessed days after treatment (Kough et al., 1987). Schreiner and Beth- species diversity were reported consistently reduced (Douds and lenfalvay (1996) investigated the effects of captan, benomyl, 12 A. WILLIS ET AL.

and PCNB on G. etunicatum, G. mosseae and Gi. rosea in pea taxa. The authors propose the animals are AM propagule vectors plants. All three depressed percentage root colonization. They not only in the terrestial habitat but also amongst high-canopy found Gi. rosea spore abundance significantly reduced in cap- epiphytes (Mangan and Adler, 2000). tan treated soils, G. etunicatum spore abundance increased in all three fungicide treated soils, and G. mosseae spore abundance D. Parasitism: The Case for Trichoderma harzianum increased only in captan-treated soils. (Ascomycota) Herbicide applications can reduce or promote mycorrhiza Green et al. (1999), in a root-free soil experiment, concluded formation. Ocampo and Barea (1985) found carbamate herbi- the biocontrol agent Trichoderma harzianum exploited the dead cides reduced soil fungal metabolism after 48 h but AM fungal mycelium and not the living biomass of G. intraradices. There root colonization levels had recovered by the end of the exper- was no adverse effect upon hyphal growth or P uptake. Their iment. Dodd and Jeffries (1989) investigated the effects of four evidence suggested T. harzianum was adversely affected by herbicides on three Glomus species in winter wheat, finding one G. intraradices. had no effect on spore germination, another prevented spore ger- Mart´ınez-Medina et al. (2011) inoculated melon seedlings mination, and the remaining two inhibited germination at low with G. intraradices, G. mosseae, and T. harzianum singly and in dosage but had a stimulatory effect at higher dosage. Glyphosate combinations. Half of the plants were inoculated with Fusarium had no effect on G. intraradices colonizing genetically modi- oxysporum after six weeks. T. harzianum alone increased shoot fied (GM) soybean (Powell et al., 2009). Simazine applications fresh wt. by 20%, neither AM fungal species increased shoot at concentrations above 1 mg kg−1 induced non-mycorrhizal fresh wt. over controls, and co-inoculated treatments fresh wt. Chenopodium quinona to form AM (Schwab et al., 1982). increase lay in-between, suggesting synergism as T. harzianum Pesticides also have variable effect on AM fungi, gener- increased AM fungal root colonization compared with Gloma- ally decreasing colonization, sometimes significantly. Abd-Alla les alone treatments. The greatest increase was observed in the et al. (2000) found significant inhibition of AM fungal root col- combination with G. intraradices. T. harzianum was unaffected onization and spore production in an investigation of the effect by either of the AM fungal species. T. harzianum alone re- of five different pesticides applied to three legume crops. Sreeni- duced disease incidence by 50%, G. mosseae also by 50%, and vasa and Bagyaraj (1989), in their assessment of the effects of G. intraradices by 25%. Disease incidence in G. mosseae - T. five insecticides on G. fasciculatum in pot culture found all were harzianum co-inoculation was no less than in individual treat- deleterious at recommended dosage but two applied at half that ments but G. intraradices - T. harzianum co-inoculation was rate significantly increased root colonization and spore density. significantly more effective than in individual treatments. The variations were correlated with decreased and increased IAA C. Grazing and ethylene levels, respectively, in stem tissue. Grazing of AM fungal spores and mycelia has been observed. Purin and Rillig (2007) surmised there is little evidence of Feeding preferences of six species of mites and collembolans, parasitism of AM fungi, suggesting glomalin may be a deterrent. among the latter Folsomia candida, were assessed in soils with They also questioned whether parasitism of AM fungi, by fungi, AM- and conidial-fungal species populations. The grazers pre- bacteria or viruses, has been conclusively demonstrated. The cri- ferred conidial fungi, and when they did graze on AM fungi they teria they described fulfilling parasitic mode, particularly fitness showed clear preference for narrower hyphae (Klironomos and parameter measurement in both populations, and confirmation Kendrick, 1996). Klironomos et al. (1999) later found fecundity of AM fungal loss of fitness in follow-up soil experiments, have Downloaded by [A. Willis] at 20:31 04 December 2012 in AM fungi-grazing F. candida severely impaired, and in F1 not been met, they said, in any enquiry. generation animals fed exclusively on AM fungi, inability to De Jaeger et al. (2010) observed invasion of in vitro Glomus produce eggs. species extraradical mycelia by T. harzianum and subsequent Atul-Nayyar et al. (2008) investigated relationships between invasion into intraradical mycelia and root cells via AM intrahy- AM fungi, alfalfa, Russian wildrye and mycophagous nema- phal growth. The mycoparasite caused protoplasm degradation. −1 todes at three levels of P fertilization (0, 20, 40 kg P2O5 ha ). In further work, again in Glomus species in vitro, De Jaeger Fungal colonization levels reduced with P fertilization with or et al. (2011) demonstrated increased P uptake by the fungus without Russian wildrye. In the presence of wildrye, lower my- but disruption of P transfer to host roots in the presence of corrhizal colonization levels concurred with higher than mono- T. harzianum. culture nematode abundance, with an almost quadruple increase of omnivorous species. They attributed reduced yield observed E. Global Climate Change (GCC) in the dual crop at low P levels compared with alfalfa monocul- The considerable literature on the effects of current global ture to enhanced nematode feeding on AM hyphae. climate change (GCC) on AM fungi is variable and in this Faeces from seven of ten small rain-cloud forest mammals, review only briefly discussed. For more comprehensive reviews and more than half of 94 samples examined, contained intact see Treseder and Allen (2000), and Treseder (2004). AM spores. Spore morphology analysis indicated the animals Predicted global surface temperature rise affects AM fungi. actively fed on AM fungi and showed preference for sporocarpic Significant increase in AM fungal root colonization and 40% ARBUSCULAR MYCORRHIZAL FUNGI 13

soil hyphal length increase in Avena barbata rhizosphere in species diversity gradient (1, 4, 9 and 16). Phospholipid fatty a soil-warming field experiment was descibed by Rillig et al. acid (PLFA) analysis indicated that rates of soil C cycling, in (2002). Compant et al. (2010), in their review of 135 studies on which AM fungi play a significant role, were reduced relative the effects of GCC on beneficial soil organisms and interaction to grasses diversity decrease. with host plants, found in the majority of investigations soil temperature increase had a positive impact on AM fungal root F. AM - AM Competition colonization and hyphal length. Competition amongst AM fungal species is difficult to assess. Rising global atmospheric CO2 levels are of concern. They All species appear so variable in form and function that it seems too affect AM fungal diversity, abundance and function. Es- impossible to draw consistent baselines from which to make sentially increase in CO2 enhances photosynthetic rates with comparisons. There is some evidence to suggest the first AM more C directed to the soil pool and AM fungi, in turn a ben- fungal taxon to colonize is frequently the most abundant within efit to plant growth. Generally AM fungi increase in hyphal the root (Hart and Reader, 2002) and that the fastest colonizers, growth and colonization but species and species-host responses e.g. Glomaceae, develop greater intraradical biomass than later are variable. Colonization in obligate mycotroph C4 grasses colonizers, e.g. Gigasporaceae, which develop greater extrarad- generally increases but not in facultative mycotroph C3 grasses ical biomass (Allen et al., 2003). Alkan et al. (2006) found G. (Tang et al. 2009). Klironomos et al. (1998) reported two Glo- intraradices and G. mosseae, as single isolates, colonized roots mus spp. colonizing Artemisia tridentata grown at 2× ambient in both directions from the point of inoculation. Where G. in- CO2 levels increased percentage arbuscular and hyphal colo- traradices colonization intensity was even, however, G. mosseae nization with significantly higher numbers of spores whereas proliferated distally. In combination both showed unidirectional an Acaulospora and a Scutellospora sp. developed significantly distal colonization. Bever et al. (2009) demonstrated preferen- higher hyphal lengths only. Johnson et al. (2005) found CO2 tial allocation of C to the more beneficial of two AM species in a elevation frequently reduced AM benefits on plant growth in a wild onion split-root system, comparing allocation of labelled- laboratory experiment. They had investigated effects on 14 plant C to roots in each of the two compartments. Adaptation to local species including forbs, C3 and C4 grasses and five AM fungal environment gives advantage over exotic species. communities, including mixtures of Glomus species and Gigas- Arbuscular mycorrhizal fungal community species richness poraceae taxa. Rillig et al. (1999; 2001b) described an increase increases with plant community diversity and is often correlated in hyphal length and consequent increase in soil concentrations with increased nutrient content and productivity in host plants of glomalin in response to rising CO2 levels. There is also some (van der Heijden et al., 1998). By inference it follows that indication increased soil microbial biomass and concomitant diversity in AM fungal functional efficiency similarly increases. increase in respiration may itself contribute to climate change Where a species is able to exploit a niche few or no other can, (Bardgett et al., 2008). traditionally a competitive edge would be ascribed, but where Anthropogenic nitrogen deposition reduces AM fungal di- does the advantage lie? The nutrient exploited, unless there is versity and abundance. Egerton-Warburton and Allen (2000) direct link with the host, is transported into a common pool assessed AM fungal diversity in scrub vegetation along an N in the mycelial web and possibly distributed amongst several deposition gradient, compared with N-fertilized and unfertil- hosts. Reciprocity may be minimal. ized plots. They found anthropogenic deposition and N exper- Individual species tolerance of, and recovery from, the effects imental plots to have similar downward trends in both abun- of fluctuation in environmental conditions might be described Downloaded by [A. Willis] at 20:31 04 December 2012 dance and diversity of AM fungal species, Gigasporaceae taxa as ‘efficiency of function’ competition. During temporarily low displaced and proliferation of small-spored Glomus species. soil-water status, for example, the species more efficient in wa- Egerton-Warburton et al. (2001) described an almost identical ter accumulation and transport to the host may increase in hy- trend, here Acaulosporaceae also displaced, in archived wood- phal length and biomass at the expense of the momentarily land soil samples sequenced with air pollution records. In a later defunct N transport (say) efficient species. However, reciprocal N fertilizer application experiment across five grasslands how- C gain should be converted to propagules, i.e., sporulation, for ever (Egerton-Warburton et al., 2007), AM fungal productivity, the species to have displayed a competitive advantage. species richness and diversity decreased in P rich soils (low N:P) The argument may have support from the rapidity of but in P limited soils (high N:P) N fertilization had an opposite AM fungal response to signaling (Drissner et al., 2007; effect. Navazio et al., 2007) and cytoplasmic streaming (Smith and There is comparatively little literature describing the effects Gianinazzi-Pearson, 1988), and evidence of AM fungal spores of elevated CO2 and N deposition interaction on AM fungi. temporal and spatial patchiness in soils (Friese and Koske, The above evidence suggests the two inputs have conflicting 1991; Carvalho et al., 2003). However, such a mechanism may influences. Klironomos et al. (1997) and Rillig and Allen (1998) perhaps be masked by variation in seasonal sporulation patterns found AM fungal hyphal length increased with CO2 in low-N but (Pringle and Bever, 2002; Escudero and Mendoza, 2005) and not in high-N treatments. Chung et al. (2007) reported increased host plant sporulation rates effects (Bever et al., 1996; Lugo CO2 effects upon soil microbial and fungal biomass over a grass and Cabello, 2002). Pringle and Bever (2002) suggested the 14 A. WILLIS ET AL.

disparate temporal sporulation niches found in two common the fungal symbiont is parasitic where it gains in nutrient ex- AM fungal species (A. colossica and Gi. gigantea)inthe change and mutualistic where net gains are equal. It is suggested grassland they studied may facilitate co-existence. here AM fungi may employ a rapid and dynamic mutualism- Other effects are reported. Preliminary investigation of the commensalism-parasitism gradient strategy whereby they con- effect of granitic rock-dust added to laterite iron-ore mine spoil tinually alter mode of action in accommodation of even the conducted by Willis and Rodrigues (unpublished) suggested in- smallest change in environmental influence. Cost : benefit ratio, crease in AM fungal spore abundance. Biochar has positive ef- to both plant and fungus, may constantly fluctuate. fects on abundance of, and colonization by, AM fungi. Warnock et al. (2007) proposed four possible mechanisms, effects on soil physico-chemical properties; effects through influence on other XVIII. CONCLUSIONS soil microbes; interactions with plant-fungus signalling, includ- Glomeromycota are complex ancient organisms, globally ing adsorption of labile and volatile toxins; and refuge from distributed and intrinsically involved in possibly all aspects of fungal grazers. Extraradical hyphal invasion of OM fraction in soil ecology, and directly or indirectly in many aspects of above- soil is often found to be extensive (Hodge et al., 2001) with ground ecology. It is clear they have considerable impact upon significant positive influence on fungal biomass, colonization their edaphic and biotic surroundings, above- and below-ground, and sporulation. Several predicted scenarios of GCC outcome spatially and temporally affecting every terrestrial niche across suggest significant increase in OM levels in soils. Where both almost every biome. Wherever there are natural plant communi- roots and fungal hyphae occupy the same OM patch however, ties, there will be AM fungal influence. Mycorrhizodeposition fungal biomass can be reduced (Hodge, 2003). A report by of photosynthates plays a fundamental role in the carbon cycle, Mack and Rudgers (2008) described a strong reduction in AM facilitating specific saprotrophic microbial community devel- fungal root colonization in the invasive grass species Schedo- opment which suggests a tripartite symbiosis in soils involving norus phoenix (tall fescue) simultaneously colonized by the plant host, AM fungi and associative microbes is common. In- symbiotic herbivore-deterring foliar endophyte Neotyphodium finite combinations of species could enhance plant growth and coenophialum. There was positive correlation between Neoty- community development in almost all spatial and temporal com- phodium hyphal density and plant biomass and negative correla- plexities of soils heterogeneity. tion with AM fungal colonization. Mycorrhizal fungi treatments The many functional roles of AM fungi and the spatial and had no significant impact on the endophyte. temporal extent of those functions at the interface between abiotic and biotic elements suggest the phylum is a primary ecosys- G. A Little P Goes a Long Way tem facilitator, a ‘keystone mutualist’ (O’Neill et al., 1991), per- Soil P scarcity is the most commonly reported effect of in- haps even a mediator in plant co-existance (Hart et al., 2003), crease in AM fungal root colonization and spore abundance, a co-operation, and self-maintaining strategies. Future research, reflection on the phylums assistance in green plants gaining a particularly quantifying AM fungal impact or “relative influ- foot-hold on dry land. Hostile, nutrient-deficient environments ence” (Klironomos et al., 2011) will help clarify these concepts, tend to carry diverse AM species even where the host plant even should, as the authors put it, “quantification raise(s) the diversity is low. Fungal species and functional diversities gen- spectre that the mycorrhizal symbiosis may not be a significant erally increase with plant community species diversity but even driver of plant community dynamics in some or many ecosys- in low P monoculture fungal diversity is generally greater than tems.” The higher plant/Glomeromycota symbiosis is, however, Downloaded by [A. Willis] at 20:31 04 December 2012 in high P status multi-host soils (Hijri et al., 2006). so entwined that it is probably implausible to study the ecology The control mechanism would seem to be direct positive of either one in isolation. feedback mediated by host plant increase in C supply. 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View publication stats RESEARCH ARTICLE Inoculation effects on root-colonizing arbuscular mycorrhizal fungal communities spread beyond directly inoculated plants

Martina JanousÏkovaÂ1*, Karol Krak1, Miroslav VosaÂtka1, David PuÈschel1, Helena SÏ torchovaÂ 2

1 Institute of Botany, The Czech Academy of Sciences, Průhonice, Czech Republic, 2 Institute of Experimental Botany, The Czech Academy of Sciences, Praha, Czech Republic

* [email protected] a1111111111 a1111111111 a1111111111 a1111111111 Abstract a1111111111 Inoculation with arbuscular mycorrhizal fungi (AMF) may improve plant performance at disturbed sites, but inoculation may also suppress root colonization by native AMF and decrease the diversity of the root-colonizing AMF community. This has been shown for the roots of directly inoculated plants, but little is known about the stability of inoculation effects, OPEN ACCESS and to which degree the inoculant and the inoculation-induced changes in AMF community Citation: Janousˇkova´ M, Krak K, Vosa´tka M, composition spread into newly emerging seedlings that were not in direct contact with the Pu¨schel D, Sˇtorchova´ H (2017) Inoculation effects on root-colonizing arbuscular mycorrhizal fungal introduced propagules. We addressed this topic in a greenhouse experiment based on the communities spread beyond directly inoculated soil and native AMF community of a post-mining site. Plants were cultivated in compart- plants. PLoS ONE 12(7): e0181525. https://doi.org/ mented pots with substrate containing the native AMF community, where AMF extraradical 10.1371/journal.pone.0181525 mycelium radiating from directly inoculated plants was allowed to inoculate neighboring Editor: Erika Kothe, Friedrich Schiller University, plants. The abundances of the inoculated isolate and of native AMF taxa were monitored in GERMANY the roots of the directly inoculated plants and the neighboring plants by quantitative real- Received: November 16, 2016 time PCR. As expected, inoculation suppressed root colonization of the directly inoculated Accepted: July 3, 2017 plants by other AMF taxa of the native AMF community and also by native genotypes of the

Published: July 24, 2017 same species as used for inoculation. In the neighboring plants, high abundance of the inoculant and the suppression of native AMF were maintained. Thus, we demonstrate that inoc- Copyright: © 2017 Janousˇkova´ et al. This is an open access article distributed under the terms of ulation effects on native AMF propagate into plants that were not in direct contact with the the Creative Commons Attribution License, which introduced inoculum, and are therefore likely to persist at the site of inoculation. permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Representative sequences are available from Introduction GenBank (accession numbers KC537331–363). By forming symbiotic association with the majority of terrestrial plants, arbuscular mycor- Funding: The study was supported by Ministry of rhizal fungi (AMF) significantly contribute to plant productivity in most ecosystems. They Education, Youth and Sports of the Czech Republic supply their host plants with hardly accessible nutrients, especially with phosphorus, and (project No. LH14285) and by the long-term research project of the Academy of Sciences of the receive, in return, photosynthetically fixed carbon. In natural conditions, roots of one plant Czech Republic (RVO 67985939). The funders had usually become colonized by many AMF species, and the plant’s benefits from the symbiosis no role in study design, data collection and depend not only on abiotic factors such as soil fertility [1], but also on the infectivity, diversity

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analysis, decision to publish, or preparation of the and composition of the root-colonizing AMF community. For example, AMF communities of manuscript. high species diversity enhance plant growth more than those of low species diversity [2]. Also, Competing interests: The authors have declared locally adapted AMF communities may be more efficient in providing plant benefits than that no competing interests exist. non-adapted AMF [3–5]. Infectivity and diversity of AMF communities is often reduced in disturbed habitats such as agroecosystems or post-mining sites [6–9]. Adding AMF propagules into soils or pre-inoculation of planted seedlings with AMF has been therefore recommended as a technology to support plant establishment and growth at these sites [10–12]. It is assumed that inoculation effects may be either positive, if the native AMF community is not sufficiently abundant or diverse to provide the maximal mycorrhizal benefits, or negligible, if the native AMF community is sufficient. This assumption, however, disregards potential negative side effects of inoculation due to the inoculants’ interactions with native AMF communities. Inoculation mostly introduces AMF genotypes that are not present at the site of inoculation, and sometimes even originate from different ecosystems or geographical regions [13]. Naturalization and spread of introduced organisms is an important topic of current biology as it may negatively affect biodiversity and ecosystem functioning [14]. Such concerns were also expressed in relation to AMF inoculations, but remain largely hypothetic due to our insufficient knowledge on AMF biogeography, population and community ecology [15]. Inoculation may substantially decrease the diversity of the root-colonizing AMF community by suppressing root colonization by native AMF [10, 16–19]. This suggests low resistance of native AMF communities against the biotic disturbance by introduction of new AMF genotypes. However, only little information is available about inoculation effects exceeding the immediate impact of propagule additions. Inoculated AMF were shown to persist in the roots of the inoculated plants two years post inoculation [10, 20–21]. We ignore, however, whether they are able to spread into root systems of newly emerging seedlings that were not in direct contact with the introduced propagules. Furthermore, we know very little about the resilience of AMF communities following inoculation-induced changes. Effects of inoculation on AMF abundances may disappear within the life cycle of a plant [22], which indicates that inoculation-induced changes in AMF diversity or community composition may be only transient, consistently with the suggestion that plants may actively promote the diversity of their symbionts [23]. On the other hand, AMF community richness may remain severely reduced throughout the whole vegetation season, and only partly restore even two years post inoculation [10]. Further time- course studies and studies employing quantitative molecular tools are therefore needed to describe the abundance dynamics of inoculated isolates and dynamics of AMF communities post inoculation. Assessing the establishment of inoculants and effects on native AMF is also complicated by considerable genetic and functional diversity encountered within AMF species [24–27]. Inocu- lation is usually performed with wide-spread species of Glomeraceae family such as Rhizopha- gus irregularis or Funneliformis mosseae [13], which are regularly present at the target sites. Inoculation then introduces new intraspecific genotypes that can possess different traits than the conspecific natives. Species-level markers document abundance of the inoculated species [28], but do not provide information about the proportion of the inoculated and native genotypes. This information, however, is essential for a complete picture of inoculation effects. In order to gain deeper insight into possible longer-term effects of inoculation into soils containing native AMF, we designed a greenhouse experiment addressing two questions: i) Is an inoculant able to spread into root systems of neighboring plants that were not in direct contact with the originally added inoculum? ii) How does inoculation affect the abundances of native AMF in the roots of the directly inoculated plants and their neighbors? The experiment was based on soil from a post-mining site and its native AMF community, i.e. on a typical

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target system for inoculations. It encompassed two host plants belonging to different functional groups in order to assess the role of host-specific factors, and two modes of inoculation —pre-inoculation of seedlings and inoculation in-situ by placing the inoculant’s propagules into soil. In addition to evaluating the AMF communities in roots, we measured the biomass of the experimental plants in order to evaluate possible changes in the plant-growth promoting effects of the AMF communities. We expected that immediate inoculation effects would be more pronounced after pre-inoculation due to priority effects [29–30] and hypothesized that the inoculant would spread into adjacent root systems, but its abundance and the impact on native AMF would decrease in comparison with the directly inoculated root systems.

Materials and methods Experimental system The experiment was based on the soil-fungi system of the coal mine spoil bank Merkur near Chomutov, North Bohemia, Czech Republic. The site was selected as a representative of a system where inoculation with AMF has been tested within the reclamation process [20, 31]. The site does not belong to any protected area nor was the study concerned with protected species. Access to the site and substrate collection were permitted by Severočeske´ doly a.s. company responsible for the reclamation of the state-owned land. Grey Miocene Clay forming the surface of the spoil bank was collected from an about 10-year-old site after removal of ruderal vegetation, and homogenized with sand in the ratio of 4:1 (v:v). The chemical parameters of the collected clay substrate were described by [31], the mixture with sand had the following main

parameters: pH(H2O) 7.5, Corg 1.15%, N 0.06%, Olsen-P (0.5 M NaHCO3 extractable) 2.93 mg kg-1. Inoculation was performed with an isolate of Rhizophagus irregularis (Błaszk, Wubet, Renker & Buscot) C. Walker & Schuessler (2010) termed ‘Chomutov’. This isolate originated from a different part of the spoil bank than the substrate used for the experimental cultivation, and had been kept in culture for about three years prior to the establishment of the experiment. It was selected, because it could be distinguished from the native R. irregularis population of the cultivation substrate by specific primers in the large subunit of mitochondrial ribosomal DNA (mtLSU, see subchapter ’Design and optimization of qPCR assays’ for details). At the same time, it was expected to be adapted to the conditions of the substrate. The experiment was performed with two host plant species: medic (Medicago sativa L. cv. Vlasta) and reed canarygrass (Phalaris arundinacea L. cv. Palaton S). Both taxa were tested for agricultural reclamation of spoil banks [31] and represent two plant functional groups (legume and grass).

Design and optimization of qPCR assays The native AMF community was characterized according to partial sequences of the large subunit of nuclear ribosomal DNA (nrLSU) as described in detail in S1 Text. This DNA region was selected due to available primers for universal amplification of all glomeromycotan taxa [32] and because it enables the design of primers for the specific amplification of species-level clades [33]. The population of the native R. irregularis clade was characterized according to partial sequences of mitochondrial ribosomal DNA (mtLSU) as detailed in S1 Text. In contrast to nrLSU, mtLSU haplotypes of R. irregularis enable to distinguish intraspecific genotypes of this species [34–35], but on the other hand, the lack of sequence information for most of the other glomeromycotan taxa makes this region unsuitable for the characterization of the whole AMF community. Thus, the effects of inoculation were followed using two primer sets: 1)

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primers based in the nrLSU targeting species-level clades of the AMF community; 2) primers based in the mtLSU discriminating between the inoculant and conspecific native genotypes. Phylogenetic analyses of the nrLSU of the native AMF community revealed five clades (S1 Fig), three of which corresponded to the species Rhizophagus irregularis (Błaszk, Wubet, Renker & Buscot) C. Walker & Schuessler (2010), Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & Schuessler 2010 and Claroideoglomus claroideum (N. C. Schenck & G. S. Sm.) C. Walker & Schuessler (2010). Primers designed previously to discriminate isolates of these three species [22, 35] were tested using plasmid standards and root samples and quantified reliably these clades of the native community. Additionally, primers were designed to specifically amplify genotypes of the other two clades of the native community, the ‘uncultured Glomeraceae’ clade and Diversispora celata clade C. Walker, Gamper & Schuessler (2009) based on the complete sequence alignment using Primer 3 Plus [36]. The preparation of plasmid standards, qPCR and the estimate of amplification efficiencies generally followed previously described steps [35]. Details on annealing temperatures, primer concentrations, amplicon lengths and the estimated amplification efficiencies for each used qPCR assay are summarized in S1 Table. Alignments of the R. irregularis mtLSU sequences revealed three haplotypes (S2 Fig) that could not be completely distinguished by specific primers. However, all of them were amplified by the primer combination GI-PH5-mtLSU-219F and GI-PH5-mtLSU-327R designed to discriminate the R. irregularis isolate PH5 from isolate ‘Chomutov’ in a previous study [35]. The latter isolate, used as inoculant in this study, was therefore amplified with the same primer combination as in the previous study [35] after confirming that no cross-amplification occurred with genotypes of the native AMF community of the spoil bank soil using plasmid standards and DNA extracts from roots. Details on the used qPCR assay are summarized in S2 Table.

Cultivation experiment Inoculum of R. irregularis ’Chomutov’ was prepared by mixing, homogenizing and air-drying substrate from four seven-month-old cultures of the isolate in a mixture of zeolite and sand (1:1) and medic (Medicago sativa) as host plant. The cultures were checked microscopically for the presence of spores and absence of contamination. The infectivity of the inoculum and the non-sterile experimental substrate (AMF substrate) were determined by the most probable number test before the experiment as previously described [22] with medic as host plant. The inoculum had an inoculation potential of 55 infective propagules (IP) ml-1 substrate, while the inoculation potential of the spoil bank substrate was only 5 IP ml-1. Plants of both species were germinated in autoclaved sand and pre-cultivated in seedling trays for three weeks in a 1:1 mixture of autoclaved zeolite and sand. One third of the seedlings of each species was inoculated with R. irregularis ’Chomutov’ during this pre-cultivation stage by amending the pre-cultivation substrate with inoculum to 30% (v:v) while the other two thirds of the seedlings were left without inoculation. Before the planting of the experiment, roots of five randomly selected inoculated seedlings per plant species were stained with trypan blue and confirmed, using a stereomicroscope, to have at least 30% of their roots colonized with the AMF. Further five randomly selected inoculated and non-inoculated seedlings per species were washed, dried at 65˚C and weighted to document possible effects of the inoculation on seedling growth. The experiment was established in plastic pots (12 × 12 × 9 cm), each divided into two equal compartments (6 × 12 × 9 cm) by a nylon mesh with mesh diameter 42 μm to exclude root competition but enable the spread of extraradical mycelium (ERM) between the

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compartments. Both compartments were filled with the mixture of spoil bank substrate and sand specified in ’Experimental system’. This substrate either contained its native AMF community (’AMF substrate’) or was sterilized by γ irradiation (’control substrate’) and both compartments contained the same substrate variant. These two substrate treatments were factorially combined with the two host plant species (M. sativa, P. arundinacea), and within each combination of plant and substrate, three inoculation treatments were established as follows. One seedling, further termed the donor (D) plant, was planted at the start of the experiment into one of the two compartments, while the second compartment remained empty at the start of the experiment. One inoculation treatment (pre-inoculation) was established with the seedlings inoculated during the pre-cultivation stage as described above. In the second inoculation treatment (in-situ), seedlings pre-grown in sterile substrate received inoculation with R. irregularis ’Chomutov’ directly at planting with inoculum mixed into the cultivation substrate of the planted compartment to 4% (v:v). The third inoculation treatment (non-inoculated) was established with seedlings pre-grown in sterile substrate and without additional inoculation. In order to decrease differences in microbial conditions among the experimental treatments, the planted compartments were irrigated with 10 ml of bacterial filtrate obtained by passing a suspension from the AMF substrate and the inoculum through filter paper (Whatman No.1). Each combination of substrate, plant and inoculation treatment was established in 12 replicates (except for the non-inoculated treatments in control substrate, which were established in six replicates only). After six weeks of cultivation, the second compartments of six pots per treatment were planted with a second plant of the same species, termed the neighboring (N) plant, without any additional inoculation. The other six pots per treatment were harvested to evaluate the growth and AMF community of the six-week-old D plants. The pots with both compartments planted were harvested after further six weeks of cultivation, i.e. when the D plants were 12 weeks old and the N plants six weeks old (Fig 1). Thus, each combination of the three factors plant species, substrate and inoculation treatment comprised three ’stages’: the six-week-old D plants, twelve-week-old D plants and six week old N plants. The only exception was the non-inoculated treatment in control substrate, which was established in six replicates only and did not include six-week-old D plants (see Fig 1). The experiment was cultivated in a glasshouse under supplemental light (12 hours, metalhalide lamps, 400 W) and fertilized once in two weeks with 50 ml of P2N3 nutrient solution per plant [37]. In some replicate pots, the N plants did not properly establish in the experimental system and died back. These replicate pots were completely excluded from the harvest and further evaluations with the consequence that replicate numbers were reduced in some treatments as specified in S12 Table.

Harvest and data collection Each root system was washed, weighed and cut into 1 cm segments. A subsample of 100 mg fresh weight was flash frozen in liquid N and stored at -80˚C. Another part was used for microscopic determination of root colonization after staining with 0.05% trypan blue in lactoglycerol [38]. The remaining roots and shoots were dried at 65˚C for 24 h and used for the determination of shoot and root dry weights. Percentage of root colonization by AMF was determined as frequency of AMF structures in root segments corresponding in length to the diameter of the microscopic field (×100 magnification; Olympus BX60). Genomic DNA was extracted from the deep-frozen samples using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. DNA extracts from root samples were quantified spectrophotometrically, and 10 ng of total genomic DNA were used as a

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Fig 1. Schematic presentation of the core experimental arrangement. At the start of the experiment (0 wks), the seedlings were either pre-inoculated (a), inoculated with propagules mixed into the cultivation substrate (b) or left without inoculation (c). After six weeks of cultivation (6 wks), six replicates of each treatment were harvested to obtain six-week-old donor plants (D6), the remaining six replicates were planted with a second seedling without any additional inoculum addition. After further six weeks of cultivation (12 wks), the harvest of the remaining replicates rendered 12-week old donor plants (D12) and six-week old neighboring plants (N6). This core arrangement was established four times: with two plant species (M. sativa and P. arundinacea), each either in substrate containing its native AMF community or in sterilized control substrate. The asterisk denotes replicates that were not established in the sterilized control substrate. https://doi.org/10.1371/journal.pone.0181525.g001

template in qPCR as previously described [36]. All harvested root systems per treatment and harvest were analyzed. All target sequences were quantified in the root samples from the AMF substrate. The abundances of the AMF taxa were calculated as copy numbers per ng of template DNA.

Data analysis and statistics Variation in root colonization was statistically analyzed for each substrate separately using three-way ANOVA with the factors plant species, inoculation and stage after arcsine transformation of the data. The non-inoculated treatment in control substrate with overall zero root colonization was excluded from the analysis. Variation in shoot biomass was assessed for each plant species and stage separately; one- way ANOVA followed by Tukey’s test was used to test for differences between each combination of substrate and inoculation treatment. The abundance (= mtLSU copy numbers) of the inoculant was statistically analyzed using four-way-ANOVA with the factors substrate, plant species, inoculation and stage. The non- inoculated treatment was excluded from the analysis. Proportion of the inoculant at the total R. irregularis population in AMF substrate, which was calculated as the sum of mtLSU copy numbers of the inoculant and the native genotypes, was analyzed by three-way-ANOVA with

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the factors plant species, inoculation and stage after arcsine transformation of the data. The same approach was used to assess the variability in the abundance (= mtLSU copy numbers) of the native R. irregularis population, but including also the non-inoculated treatment into the analysis. The composition of the AMF community in AMF substrate was assessed based on nrLSU copy numbers of the species-level clades without distinguishing between the inoculant and native genotypes within the R. irregularis clade. As all samples contained the same four AMF taxa, the composition of the AMF community was described by Pielou’s evenness index. The index was calculated based on Shannon’s diversity index as J’ = H’/H’max, where H’ is Shan- non’s diversity index and H’max = ln (4). The variability in J’ and in the abundance of each taxon was evaluated by three-way ANOVA with the factors plant species, inoculation and plant stage. The abundances of the other AMF taxa than R. irregularis were also summed to ’abundance of the non-inoculated taxa’, which was analyzed in the same way. The ANOVAs were calculated in STATISTICA (version 12, StatSoft, Inc., USA). When necessary, the abundance data were square-root or logarithmically transformed to fulfill the assumption of ANOVA on homogeneity of variance (tested by Levene’s test). Multiple comparisons were performed using Tukey’s test. Additionally, AMF community composition was analyzed by multivariate analyses in CANOCO [39] (version 5.0). Redundancy Analysis (RDA) was performed with non-transformed data ’centered and standardized by species’. The significance of the effects was tested using a Monte Carlo test with 499 permutations.

Accession numbers Representative sequences of partial nrLSU of the native AMF taxa were submitted to GenBank under the accession numbers KC537331–360. Partial mtLSU sequences of native R. irregularis haplotypes were submitted under the accession numbers KC537361–363.

Results Root colonization by AMF In AMF substrate, root colonization ranged between 52% and 83% (average value per treatment, S3 Table) and was significantly higher in P. arundinacea than in M. sativa. Root colonization was also affected by the factor stage and the interaction of stage and inoculation (S4 Table) in AMF substrate, but there were no significant differences among the inoculation treatments within each stage. No root colonization was found in the non-inoculated plants in control substrate. The inoculated plants in control substrate had overall higher root colonization than plants growing in

AMF substrate (F(1,168) = 19.619, P < 0.001). Similarly as in AMF substrate, root colonization was significantly higher in P. arundinacea than in M. sativa, and affected by the factor stage and the interaction of stage and plant species; inoculation had no effect, i.e. the pre-inoculated and in-situ inoculated plants did not significantly differ in root colonization (S4 Table).

Establishment of the inoculant and response of native R. irregularis genotypes The abundance of the inoculant R. irregularis Chomutov, determined as mtLSU copy numbers, was significantly higher in control substrate than in AMF substrate; the significant interaction of the factors substrate and plant species reflects that the difference was more

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Fig 2. Proportion of inoculated R. irregularis Chomutov at the R. irregularis population. Triangles and circles refer to the pre-inoculated and in-situ inoculated treatment, respectively. D6 and D12 are directly inoculated donor plants harvested after six or 12 weeks of cultivation, respectively; N6 are six-week-old neighboring plants. Data are pooled for the two plant species M. sativa and P. arundinacea, each symbol represents the mean of 9±12 replicates (± SD), for exact replicate numbers see S12 Table. Means significantly different at P < 0.05 according to Tukey's test are marked by different letters. https://doi.org/10.1371/journal.pone.0181525.g002

pronounced in P. arundinacea than in M. sativa (S5 and S7 Tables). Inoculation or stage had no effect on the abundance of the inoculant (S5 Table). In the inoculated treatments in AMF substrate, the proportion of the inoculant at the total R. irregularis population ranged between 76 and 92% (average per treatment). The proportion was significantly higher in M. sativa than in P. arundinacea and varied among the stages depending on the form of inoculation (Fig 2, S6 Table). After in-situ inoculation, the proportion was significantly higher in 12-week-old than in six-week-old D plants, but it did not differ between 6-week-old D and N plants. The abundance of the native R. irregularis genotypes was significantly affected by inoculation, stage and the interaction of both factors (S6 Table). In-situ inoculation and pre-inoculation significantly decreased their abundance throughout the experiment as compared to the non-inoculated treatment; the interaction consisted in the varying size of the effect (Fig 3, S7 Table).

Effect of inoculation on the species-composition of the AMF community The Diversispora celata clade could not be detected by the specific qPCR system in any of the experimental samples. The remaining four species-level clades were found in all root samples from AMF substrate so that the species richness of the root-colonizing AMF community was unaffected by inoculation. However, inoculation significantly affected the evenness index J’ of the community, the effect depended on stage and plant species (S8 Table). Specifically, pre- inoculation and in-situ inoculation decreased J’, as compared to the non-inoculated treatment, in the D plants of both plant species, while J’ was unaffected by inoculation in the N plants of M. sativa (Fig 4A) and significantly decreased by pre-inoculation in the N plants of P. arundinacea (Fig 4B). The effect of inoculation on community evenness J’ was mainly due to more pronounced dominance of R. irregularis in the inoculated treatments. Inoculation generally increased the

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Fig 3. Abundance of native R. irregularis genotypes and the inoculant R. irregularis Chomutov. Black boxes and empty boxes show copy numbers of mitochondrial ribosomal DNA of the native genotypes and the inoculant, respectively, in the roots of plants cultivated without inoculation (NI), inoculated in-situ (in-situ) or preinoculated (pre) with R. irregularis 'Chomutov'. D6 and D12 are directly inoculated donor plants harvested after six or 12 weeks of cultivation, respectively; N6 are six-week-old neighboring plants. Data are pooled for the two plant species M. sativa and P. arundinacea, each box is the mean of 9±12 replicates (± SD), for exact replicate numbers see S12 Table. Letters refer to the black boxes, means significantly different at P < 0.05 according to Tukey's test are marked by different letters. https://doi.org/10.1371/journal.pone.0181525.g003

abundance of R. irregularis and decreased the sum of abundances of the other AMF taxa. The effect of inoculation, however, depended on plant stage and, in the case of the other AMF taxa, also on plant species (S8 Table). To summarize comparisons among inoculation treatments performed for each stage separately (Fig 5), inoculation mostly affected the abundance of R. irregularis and the non-inoculated taxa in D plants and mostly had no effect in N plants. The abundances of each of the non-inoculated AMF taxa, F. mosseae, C. claroideum and the uncultured Glomeraceae clade, and their responses to the experimental factors are given in S9 and S10 Tables. The variation in AMF community composition data was explained by the three experimental factors by 24.5% (pseudo F = 7.4, P = 0.002). Inoculation accounted for the highest proportion of the explained variation (15.6%), followed by stage (6%) and plant species (4%). RDA with the factor plant species as covariate confirmed, in separate models, significant effects of inoculation (pseudo F = 10.9, P = 0.002), stage (pseudo F = 4.8, P = 0.004) and interaction of the two factors (pseudo F = 4.1, P = 0.002). Model including both factors and their interaction (pseudo F = 6.6, P = 0.002), with plant species as covariate, revealed close association of R. irregularis with the pre-inoculated treatment and similarity of the AMF communities after pre- inoculation in all three stages (Fig 6). In contrast to the pre-inoculated treatment, the composition of AMF communities without inoculation was variable in the three plant stages, with relatively high abundance of F. mosseae in six-week old D plants and the uncultured Glomeraceae clade in 12-week old D plants.

Plant growth At the time of their planting into the experiment, the pre-inoculated seedlings of P. arundinacea were significantly smaller than the non-inoculated seedlings (t = -2.97, P = 0.02, S11 Table). The same trend, marginally non-significant, was observed in M. sativa (t = -2.20, P = 0.06, S11 Table). In the experiment, M. sativa had the smallest biomass when non-

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Fig 4. Pielou's evenness index J' of the arbuscular mycorrhizal fungal communities. Roots of (A) M. sativa and (B) P. arundinacea, each either without inoculation (empty circles), inoculated in-situ (full circles) or pre-inoculated (full triangles) with R. irregularis. D6 and D12 are directly inoculated donor plants harvested after six or 12 weeks, respectively; N6 are six-week old neighboring plants. Each symbol represents the mean of 4±6 replicates (± SD), for exact replicate numbers see S12 Table. Means significantly different at P < 0.05 according to Tukey's test are marked by different letters. https://doi.org/10.1371/journal.pone.0181525.g004

mycorrhizal, i.e. in the non-inoculated treatment in control substrate (Table 1). Six-week old D plants of M. sativa were significantly smaller after in-situ inoculation than after pre-inoculation or with native AMF only. Twelve-week old D plants of M. sativa had the highest biomass when grown pre-inoculated in control substrate, and the biomass of N plants did not differ among the experimental treatments except for the smallest biomass of the non-mycorrhizal plants. P. arundinacea plants grew better in control substrate than in AMF substrate (Table 1). The biomass of D plants was unaffected by inoculation treatment in either substrate, N plants had the highest biomass when non-mycorrhizal (non-inoculated in control substrate) or when pre-inoculated in control substrate.

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Fig 5. Abundance of each taxon within the arbuscular mycorrhizal fungal communities. (A) Roots of M. sativa, (B) roots or P. arundinacea, each either without inoculation (NI), inoculated in-situ (in-situ) or pre- inoculated (pre) with R. irregularis. Boxes show copy numbers of nuclear ribosomal DNA, empty boxes refer to R. irregularis, the other, non-inoculated taxa are distinguished by colors (F. mosseaeÐblue, C. claroideum Ðgreen, uncultured GlomeraceaeÐyellow). D6 and D12 are directly inoculated donor plants harvested after six or 12 weeks, respectively; N6 are six-week old neighboring plants. Boxes are means of 4±6 replicates, for exact replicate numbers see S12 Table. Vertical lines show SD for the abundance of R. irregularis or the sum of abundances of the non-inoculated AMF taxa. Asterisks refer to the same values as the SD and indicate, within D6, D12 or N6, significant difference from the non-inoculated treatment at P < 0.05 according to Dunnett's test. https://doi.org/10.1371/journal.pone.0181525.g005

Discussion This study presents, for the first time, experimental evidence for inoculation effects exceeding the directly inoculated plants. We found that 1. The inoculant spread into the root systems of neighboring plants; 2. The suppression of native AMF by inoculation, observed in the directly inoculated plants, was maintained in the neighboring plants.

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Fig 6. Redundancy analysis of the arbuscular mycorrhizal fungal communities. The model (pseudo F = 6.6, P = 0.002) included the experimental factors inoculation and stage and their interaction; plant species was added as covariate. It accounted for 36.8% of the variability in the data (22.7% is explained by the first axis and 11.2% by the second axis). Black arrows represent taxa of the arbuscular mycorrhizal fungal communities (RIÐR. irregularis, FMÐF. mosseae, CCÐClaroideoglomus claroideum, UNÐuncultured Glomeraceae), black circles are inoculation treatments (NIÐnon-inoculated, insÐinoculated in-situ, preÐ pre-inoculated), black squares are stages (D6±6-week-old donor plants, D12±12-week-old donor plants, N6 Ðneighboring plants), triangles in colors are combinations of inoculation and stage. https://doi.org/10.1371/journal.pone.0181525.g006

Establishment of the inoculant Establishment of inoculated AMF in the roots of directly inoculated plants, observed in accordance with many previous studies, e.g. [10, 17, 20, 28], indicates that the inoculant successfully competed with native AMF in primary infection, intraradical spread and the formation of secondary infection units. This is facilitated by the inoculant’s priority, either directly achieved by pre-inoculation or supported by high inoculum doses [23, 40], and does not necessarily mean establishment of a stable population of the inoculant at the site of inoculation. However, propagation of the inoculant into neigboring root systems, as documented in our experiment, requires also competitiveness in the spread of ERM and formation of infective propagules in soil. During these later stages of the symbiosis, the highly abundant inoculant may lose its competitiveness e.g. due to sanctioning by the host plant through preferential allocation of carbon to other AMF [41]. The comparable abundance of the inoculant in the directly inoculated plants and their neighbors of our experiment, however, do not indicate such a mechanism. Inoculation did not introduce a new species in our experiment, as R. irregularis was also part of the native community. However, it introduced a new intraspecific genotype, which

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Table 1. Shoot dry weights of M. sativa and P. arundinacea.

Factors Experimental Stage Plant Substrate Inoculation D6 D12 N6 M. sativa Control NI n.d. 0.88 (0.38) c 0.13 (0.05) b in-situ 0.30 (0.08) b 2.01 (0.30) b 0.43 (0.11) a pre 0.76 (0.09) a 2.71 (0.37) a 0.50 (0.16) a AMF NI 0.93 (0.12) a 1.95 (0.39) b 0.49 (0.08) a in-situ 0.42 (0.07) b 1.64 (0.27) b 0.55 (0.19) a pre 0.75(0.11) a 1.81 (0.16) b 0.56 (0.09) a df 4 5 5 F-value 41.2 *** 17.3 *** 8.4 *** P. arundinacea Control NI n.d. 2.74 (0.40) a 1.18 (0.19) a in-situ 0.86 (0.22) ab 2.76 (0.29) a 0.38 (0.09) b pre 1.16 (0.20) a 2.80 (0.18) a 0.89 (0.32) a AMF NI 0.46 (0.21) c 1.08 (0.41) b 0.46 (0.20) b in-situ 0.56 (0.18) bc 0.70 (0.20) b 0.36 (0.19) b pre 0.41 (0.11) c 0.97 (0.22) b 0.30 (0.16) b df 4 5 5 F-value 14.1 *** 52.3 *** 13.7 ***

The plants were grown in substrate with native AMF community (AMF) or sterilized control substrate (Control), non-inoculated (NI), inoculated in-situ (in- situ) or pre-inoculated (pre) with R. irregularis `Chomutov'. D6 and D12 are directly inoculated donor plants harvested after six or 12 weeks of cultivation, respectively; N6 are six-week-old neighboring plants. Values are g of shoot dry biomass, means of 4±6 replicates (SD), for exact replicate numbers see S12 Table. F-values are given according to one-way ANOVA with substrate × inoculation combination as factor. *** P < 0.001. Means within column significantly different at P < 0.05 according to Tukey's test are marked by different letters; n.d. not determined. https://doi.org/10.1371/journal.pone.0181525.t001

may ecologically and functionally have differed from the native genotypes [24, 42–43] and may have remained a separate genetic entity in case of vegetative incompatibility with the native genotypes [44–45]. The naturalization of an introduced organism occurs when it crosses the barrier of reproduction [46], and we argue that due to the obligate biotrophy of AMF, their successful reproduction is demonstrated by the ability to infect plants that were not in direct contact with the introduced propagules. In this respect, our experiment demonstrated naturalization of an AMF inoculant and establishment of a stable population, albeit in the artificial conditions of pot cultivation. Moreover, the documented horizontal spread by below-ground hyphal growth constitutes a vector of AMF dispersal [47]. Thus, the ability to infect neighboring plants by ERM indicates also dispersion potential, at least at small-scale level.

Inoculation effects on native AMF Concordantly with our results, inoculation was previously shown to increase the abundance of the inoculated AMF species and to reduce or even completely eliminate root colonization by other AMF species [10, 17–18, 28]. No complete exclusion of native AMF in the inoculated treatments of our experiment should be attributed to the employed detection system: The characterization of the AMF community was performed by Sanger sequencing of clones and certainly missed some low abundant taxa [48], which may also partly explain the relatively low diversity of the native community. However, the sensitivity of qPCR for low abundant taxa is even lower, as indicated by the failure to detect the D. celata clade. Thus, the employed approach concentrated on the relative abundances of the more abundant AMF community members, which may be expected to have larger impact on the functioning of the symbiosis.

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Overdominance of one species occurs also in natural AMF communities and seems revers- ible by stochastic effects [49–50]. However, we cannot exclude that overdominance induced by inoculation may be permanent or even progressive and negatively impact on the functional parameters of the AMF community [51]. In our experiment, the inoculated and non-inoculated AMF communities became more similar in the neighboring plants than in the directly inoculated plants. However, this was rather due to shifts in the non-inoculated community than to changes in the inoculated communities that remained similar across the three stages. Therefore, we did not find any convincing evidence for short-term resilience in the AMF community composition after inoculation. This may be attributed to the specific conditions of the experiment: The tendency to increasing dominance of R. irregularis in the non-inoculated treatment indicates that this species was favored by the cultivation conditions and the effect of inoculation partly anticipated a trend that occurred also in the non-inoculated community. On the other hand, the presumably suitable conditions may also have progressively favored the inoculant alongside with progressive suppression of the other species, which was neither the case. The effect of inoculation was stable in the investigated time-frame, the AMF communities of the inoculated treatments were similar in the directly inoculated plants and in their neighbors. A previous study suggesting short-term resilience of AMF communities after inoculation differed from the presented experiment in having inoculated the same intraspecific genotypes as already present in soil [22]. New genotypes may introduce new traits into a species’ population and thus alter the population dynamics and the species’ competitiveness within the AMF community. Additionally, genetically different conspecific isolates compete among each other [52], which is consistent with the abundance decline of native R. irregularis genotypes in the inoculated treatments of our experiment. Cultivated isolates of R. irregularis usually contain only one mtLSU haplotype [53–54], while mtLSU diversity is considerably higher at field sites [34, 55], probably due to cultivation-related bottlenecks and biases [34]. Consistently, we found only one mtLSU haplotype in the inoculated R. irregularis isolate, which had been long- term maintained in culture, and three haplotypes in the root-colonizing native population. Inoculation thus, on one hand, increased the genotype richness of the R. irregularis population by introducing a new genotype, but on the other hand probably also decreased its evenness by suppressing the genetically more diverse native population. Thus, the effects of inoculation were analogous at the interspecific and intraspecific level. However, we must be aware that intraspecific interactions are more diverse than interspecific interactions including also possible anastomosis formation and genetic exchange between genotypes [44–45]. Understanding them more in detail would require screening the populations with multilocus markers [56]. Yet, mtLSU haplotypes also represent functionally relevant categories and, in contrast to multilocus screenings, can be directly detected and quantified in experimental samples [34–35]. The pattern of inoculation effects was largely consistent between the two plant species and two modes of inoculation At community level, however, pre-inoculation tended to suppress the native AMF more than inoculation in situ (Fig 6). Different impact of the two modes of inoculation was anticipated due to previously documented suppression of colonization in roots already colonized by another AMF species [29–30, 57]. Interestingly, the differences between the pre-inoculated and in-situ inoculated AMF community persisted also in the neighboring plants. This indicates that priority effects are maintained also at the level of ERM infectivity, possibly by ensuring better excess to carbohydrates from the host plant [29, 57]. The selected system and the experimental approach strongly suggest that the native AMF community was composed of disturbance-tolerant ruderal AMF, which is also consistent with the phylogenetic placement of all the detected clades in Glomerales order [58]. It can be assumed that competition between the inoculant and the native community was more intense

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than if they were phylogenetically and functionally more distinct [2, 59]. However, ruderal systems such as early successional areas and fields are typical target sites of AMF inoculations, and inoculation is typically performed with easily cultivable, i.e. fast growing isolates originating from similar conditions as the target system [13]. For this reason, we may assume that the intensity of competition and shifts in AMF community composition encountered in our study are representative for systems where inoculation is usually performed.

Functional impact of inoculation Comparing the growth of non-mycorrhizal plants (i.e. non-inoculated plants in control substrate) and mycorrhizal plants (in all the other treatments) suggests that the two selected plants species differed in their response to mycorrhiza. The positive mycorrhizal growth response of M. sativa is consistent with some previous studies, e.g. [22, 60, 61], but its magnitude should not be overinterpreted in view of other possible microbial factors, which may have contributed. Despite the addition of microbial filtrates, rhizobial population was probably initially less abundant in the non-inoculated control substrate than in the other treatments, which may have influenced M. sativa growth along with mycorrhiza. In contrast to M. sativa, P. arundinacea growth throughout the experiment indicates accumulation of pathogens in the experimental system, which was especially harmful to the later planted N seedlings. Nevertheless, these considerations on the overall response to mycorrhiza in the two plant species do not discard comparisons of plant growth among the different inoculation treatments in AMF substrate. There, the microbial community was dominated by the microorganisms introduced with the original spoil bank soil, which constituted 80% of the cultivation substrate. Positive effects of inoculation are usually related to higher root colonization [62]. Hence, it is not surprising that inoculation did not enhance plant growth in the AMF substrate, where the native AMF community alone ensured high root colonization levels. In contrast, growth of M. sativa was reduced by inoculation in-situ in comparison to both the non-inoculated and pre-inoculated treatment. It has been suggested that inoculation in-situ may suppress plant growth by increasing competition among the root-colonizing AMF [22]. In the roots of pre- inoculated plants, fungal competition may be less intense as further root colonization of already mycorrhized plants is regulated prior to AMF-root contact, via signals in root exudates [63]. However, in-situ inoculated M. sativa plants produced less biomass than pre-inoculated plants also in the control treatment with no native AMF. This suggests, alternatively, that the inoculant imposed high initial carbon costs on the host plant prior to starting supplying it with nutrients [64–65], which is consistent with the effect of pre-inoculation on seedling growth in the pre-cultivation stage. Altogether, plant growth did not indicate any inoculation-induced changes in the symbiotic efficiency of the AMF community, reported in some previous studies [17, 66]. However, our data suggest that pre-inoculation is more suitable to immediately promote plant growth than inoculation in-situ, possibly also because it suppresses interactions among the inoculant and native AMF during the root colonization process.

Conclusions and outlooks Propagation of inoculants and inoculation effects exceeding directly inoculated plants had been previously assumed both in relation to desired long-term improvement of symbiotic AMF communities [13, 67] and to possible negative consequences of inoculations [15], but a direct proof was missing. Our work represents the first experimental evidence for the spread of inoculated AMF and inoculation effects beyond the directly inoculated plants. The main result was that root colonization by native AMF was similarly suppressed in the neighboring plants as in the directly inoculated plants. This was largely independent of host

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plant and inoculation procedure. Effects of inoculation on AMF diversity and mycorrhiza function, however, also depend on the inoculant’s identity [10, 17, 19], which had not been manipulated in our experiment. Further studies should therefore focus this factor in order to confirm to which degree the findings depend on the inoculant’s taxonomic identity and/or functional traits. The employed cultivation system is suitable for such a screening as it enables the elimination of interfering factors such as space heterogeneity of native AMF communities and climatic fluctuations. On the other hand, our experiment has not supported the idea of short-term post- inoculation resilience of AMF communities, and therefore shifts attention to long-term dynamics, which should be preferentially targeted in field conditions. Both directions must be followed in order to further improve our understanding of inoculation effects, which is critically needed for assessing the potential benefits and drawbacks of AMF inoculations.

Supporting information S1 Text. Characterization of the diversity of native arbuscular mycorrhizal fungi. (PDF) S1 Fig. Phylogenetic analysis of the native arbuscular mycorrhizal fungal community. (PDF) S2 Fig. Phylogenetic analysis of Rhizophagus irregularis haplotypes. (PDF) S1 Table. Real-time PCR assays in nuclear ribosomal DNA. (PDF) S2 Table. Real-time PCR assays in mitochondrial ribosomal DNA. (PDF) S3 Table. Variation in root colonization. (PDF) S4 Table. Root colonization of the experimental plants. (PDF) S5 Table. Variation in the abundance of the inoculant R. irregularis Chomutov. (PDF) S6 Table. Variation in the proportion of the inoculant and the abundance of native R. irregularis. (PDF) S7 Table. Abundances of native R. irregularis and inoculated R. irregularis Chomutov. (PDF) S8 Table. Variation in Pielou’s evenness index J’, abundance of Rhizophagus irregularis and the sum of abundances of other AMF taxa. (PDF) S9 Table. Abundances of C. claroideum, ’uncultured Glomeraceae’ and F. mosseae. (PDF) S10 Table. Variation in the abundances of C. claroideum, ’uncultured Glomeraceae’ and F. mosseae. (PDF)

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S11 Table. Total dry weights of M. sativa and P. arundinacea D seedlings at planting into the experiment. (PDF) S12 Table. Numbers of evaluated replicates per experimental treatment. (PDF)

Acknowledgments We are grateful to Jana Marsˇ´ıčkova´ for her excellent technical assistance and to Petr Kohout for useful comments on an earlier version of the manuscript.

Author Contributions Formal analysis: MJ KK. Funding acquisition: MJ MV HSˇ. Investigation: MJ KK DP. Methodology: HSˇ KK. Resources: DP KK HSˇ. Writing – original draft: MJ KK. Writing – review & editing: MJ KK HSˇ MV DP.

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