1 INTRODUCTION There is a global industry built upon the production of “bioinoculants,” which includes arbuscular mycorrhizal fungi (AMF). These fungi establish symbiotic associations with the roots of most plants and are known to improve plant performance. Currently, only a few AMF genotypes are produced as bioinoculants and they are distributed globally, but evidence for their efficacy is scant, incomplete or lacking altogether. Perhaps more troubling is that the scientific community has little idea of the ecological consequences of the use of AMF inocula. This is a clear case where industrial practices are not aligned with current scientific knowledge. The ethical and economic implications of such oversight are profound, and ranges from customer quality assurance to soil biodiversity and ecosystem functioning. Here, we outline important gaps in knowledge, both in terms of inoculant efficacy and ecosystem integrity.

2 A RECURRING REVOLUTION Every 10 years or so, the idea of using AMF as a bioinoculant is rediscovered (e.g. Abbott & Robson, 1982; Hart & Trevors, 2005; Mcgonigle, 1988; Rodriguez & Sanders, 2015). The story is similar in every iteration: AMF are powerful tools that will improve yields and reduce inputs. These claims are based on the growing body of knowledge clearly showing that AMF help plants survive stressful growing conditions (Lenoir, Fontaine, & Lounès‐Hadj, 2016; Liu et al., 2007). However, unlike other bioinoculants (i.e. rhizobia), there is yet little proof that inoculation by commercial AMF is useful in cropping systems. Are increases in yield sufficient to offset the added expense of inoculum application? Are the results consistent/predictable? While the idea of increasing agricultural yield without concomitant increases in fertilizer is appealing environmentally, there are many unknowns about these products. Despite the reported variability in plant growth promotion following inoculation with AMF (Hoeksema et al., 2010), a growing number of companies world‐wide produce and market AMF inoculants (Dalpé & Monreal, 2004) (Figure 1). There is no universally adopted code of “best practice” regarding AMF inoculum selection 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 spiked the optimism of inoculant producers. Whatever the reason, the burgeoning inoculant market has flourished in the absence of adequate risk assessment or global regulatory policies on their production and distribution (Malusá & Vassilev, 2014; Owen, Williams, Griffith, & Withers, 2015; Vosátka, Látr, Gianinazzi, & Albrechtová, 2012). Figure 1 Open in figure viewer PowerPoint 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 Studies examining arbuscular mycorrhizal fungi (AMF) bioinoculants conducted by country from 1970 to 1999.= 193 (a), and 2000 to 2014,= 444 (b). Inoculum producers by country that cited in both (a) and (b). Countries producing inoculum that were cited in (a) and (b),= 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 If previous attempts to incorporate AMF inoculants into mainstream agrosystems were stalled by lack of research tools (Hart & Trevors, 2005; Schwartz et al., 2006), this is no longer an obstacle. Molecular advances in the last decade now make it possible to track inoculation of single genotypes (Farmer et al., 2007; Hart et al., 2013; Jansa, Smith, & Smith, 2008; Rodriguez & Sanders, 2015). However, despite the ability to assess the abundance of inoculants in the environment, the effectiveness and risk of AMF inoculation remain unclear.

4 EVIDENCE FOR IMPROVED PLANT RESPONSE Meta‐analyses indicate that growth responses to AMF inoculation are, on the whole, inconsistent (Hoeksema et al., 2010; Lekberg & Koide, 2005; Mcgonigle, 1988; Pellegrino, Öpik, Bonari, & Ercoli, 2015). Part of the reason for inconsistency within these analyses stems from the fact that both field and greenhouse studies were considered together, but high variation persists even among field studies. A spate of recent studies show an increase in plant biomass post inoculation (e.g. Camprubi, Zárate, Adholeya, Lovato, & Calvet, 2015; Cely et al., 2016; Hijri, 2016; Pellegrino et al., 2012; Tawaraya, Hirose, & Wagatsuma, 2012). Most of these studies, especially those evaluating multiple plant taxa, either show positive responses to inoculation (e.g. Abbott & Robson, 1982; Abbott, Robson, & Hall, 1983; Antunes et al., 2009; Buysens, César, Ferrais, Dupré de Boulois, & Declerck, 2016; Caravaca, Alguacil, Barea, & Roldán, 2005; Ceballos et al., 2013; Douds, Wilson, Seidel, & Ziegler‐Ulsh, 2016; Emam, 2016; Middleton et al., 2015) or no effect at all (e.g. Farmer et al., 2007; Pellegrino & Bedini, 2014). Studies showing reductions in plant performance following inoculation have also been reported (e.g. Aprahamian et al., 2016; Herzberger, Meiners, Towey, Butts, & Armstrong, 2015; Janoušková et al., 2013; Verbruggen, Kiers, Bakelaar, Röling, & van der Heijden, 2012). However, few studies measure plant reproductive output, yield or other variables that correspond to meaningful gains in cropping systems. 4.1 Partner identity Variation in host response to inoculation may be partly explained by the identity of the host and fungus. For example, host response to inoculation can vary with cultivar (Buysens et al., 2016; Ceballos et al., 2013; Douds et al., 2016; Pellegrino et al., 2015). Similarly, fungal provenance can lead to different inoculation outcomes: positive inoculation 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 (Caravaca et al., 2005; Davidson, Novak, & Serpe, 2016; Emam, 2016; Labidi et al., 2015; Maltz & Treseder, 2015; Middleton et al., 2015; Pellegrino & Bedini, 2014; Rúa et al., 2016). Given that the efficacy of the AM symbiosis is influenced by soil fertility (Tawaraya et al., 2012), inoculation timing (Mummey, Antunes, & Rillig, 2009), site disturbance level (Antunes et al., 2009) and, perhaps partner co‐adaptation (Koyama, Pietrangelo, Sanderson, & Antunes, 2017; Rúa et al., 2016), it is not surprising that it is difficult to detect a uniform host response to inoculation. This does not bode well for predictable commercial inoculation outcomes. 4.2 Inflated positive outcomes Is the surfeit of recent, positive outcomes applicable to real world scenarios? Most inoculation studies are conducted under artificial greenhouse conditions which do not inform performance in the field (Faye et al., 2013; Ohsowski, Zaitsoff, Öpik, & Hart, 2014). Furthermore, biases within field studies may be more pernicious, as they are more subtle than those in greenhouse studies. 4.2.1 Degraded systems The majority of field inoculation studies have been conducted in severely degraded soils (i.e. restoration post mining) or soils with very low inoculum potential (e.g. some situations in conventional agriculture or desertified landscapes). Extreme conditions increase the probability of detecting a positive inoculation outcome because propagule abundance is tightly linked to mycorrhizal response (Allen & Allen, 2005). Thus, AM plants should respond positively to inoculation when growing in an inoculum‐limited environment (Abbott & 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 resident AMF communities are well‐established, as in most agricultural soils (Oehl et al., 2010). Furthermore, custom inocula sourced from reference ecosystems have been shown to perform better than commercial inocula in restoration campaigns (Maltz & Treseder, 2015). To balance knowledge of inoculant performance, it will be important to conduct inoculation experiments across a broad range of soil conditions, including different land use types, and intact ecosystems to better predict where inoculation may be effective. Understanding the local conditions is essential for predicting success of inoculation as the absence of AMF may reflect unsuitable soil conditions or land management practices that may preclude establishment and persistence of inoculant AMF. 4.2.2 Inappropriate controls Erroneous comparisons can result when inoculated plants are compared to non‐inoculated controls (Boyer, Brain, Xu, & Jeffries, 2015; Burkle & Belote, 2015). For example, where plants must first be propagated in nurseries prior field transplantation, uninoculated “controls” typically perform more poorly than inoculated plants (e.g. Camprubi et al., 2015; Hernádi, Sasvári, Albrechtová, Vosátka, & Posta, 2012; Tawaraya et al., 2012). In this situation, control plants are deprived of the AM symbiosis during critical developmental stages, and do not provide a valid control. While this approach may be relevant for transplanted horticultural crops (i.e. grapevines and some field vegetables), in general, the existence of AMF‐free plants is highly unlikely (Lekberg & Koide, 2014). Thus, using uninoculated “controls” in systems requiring transplants does not represent a valid test of inoculation response.

5 EVIDENCE FOR SUCCESSFUL ESTABLISHMENT? In most field studies, inoculant establishment is not reported. Without evidence of inoculant establishment, responses are merely correlative, and it is not possible to ascribe changes in host performance to the introduction of AMF inocula (Rodriguez & Sanders, 2015). If inoculation results in a growth response in the absence of inoculant establishment, can we say that inoculation is “successful?” Unless carefully controlled for, observed changes (positive or negative) maybe due to concurrent “fertilizer/amendment effects” potentially associated with inoculants, such as the co‐introduction of other microbiota with the inoculant carrier, inclusion of nutrients or simply due to inoculation protocols, rather than the AMF in the inoculant itself. 5.1 Root colonization While root colonization is commonly reported in studies of responses to inoculant AMF, this information reflects inoculant establishment only in systems with no resident fungi. It has little value in field trials because even in extremely degraded soils, it is difficult to distinguish among inoculants and fungi naturally dispersing into the site (Bell, Wells, Jasper, & Abbott, 2003). In such conditions, it can at best indicate an indirect influence, such as through changes in the AMF community (Rodriguez & Sanders, 2015). 5.2 Tracking specific inoculants Attempts to directly track inoculants are few, thus knowledge of inoculum establishment and persistence is scant. High variability reported in inoculum detection may be due to the inability of current marker genes to effectively distinguish among closely related taxa. This is particularly the case for commercial inoculants, which comprise cosmopolitan taxa, as the current barcoding gaps in ITS, LSU and 18S are unable to distinguish among isolates within a species (Hart et al., 2015). Approaches based on isolate‐specific targets offer greater promise but are less common. Again, studies using this approach have shown variable outcomes (Farmer et al., 2007; Symanczik, Courty, Boller, Wiemken, & Al‐Yahya'ei, 2015). While Köhl, Lukasiewicz, and 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 et al., 2012) and 3 years (Sykorova et al. 2015), but these studies also reported inconsistent establishment. Long‐term inoculation trials are clearly needed, but confirmation of inoculant establishment will remain a challenge due to the inherent complexity of AMF genetic organization. AMF are known to contain multiple haplotypes per isolate (Koch et al., 2004), which may lead to under‐estimation of the inoculant if all haplotypes are not targeted by the approach. Within species, AMF strains can anastomose, which could lead to both an under‐estimation of the inoculant fungi and a gradual dilution of effects (De La Providencia, De Souza, Fernández, Delmas, & Declerck, 2005). Furthermore, DNA can persist in the environment in quiescent and dead cells (Levy‐Booth et al., 2007) leading to the detection of inoculants that have failed to establish. So what can we say about inoculant establishment? (1) Early establishment appears to be highly variable and (2) there is little evidence that inoculants persist. Considering that, on average, only 10% of all exotic species are able to establish and persist in a novel habitat (Williamson, 1996; also, see Figure 2 and Box 1 for What drives inoculant spread?), it may be difficult to select an AMF inoculant that persists between growing seasons. Whether this is a concern for end users is not yet clear. It may not be necessary for inoculants to persist in the soil if inoculation benefits can be achieved by repeated inoculation. However, knowledge of both the establishment and persistence of commercial AMF inoculants (Abbott, Robson, & Gazey, 1992) is important in order to properly assess cost/benefit analysis and to develop best practice protocols (Abbott & Lumley, 2015). Figure 2 Open in figure viewer PowerPoint 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 Box 1. What determines if an inoculant will spread? (Associated with Figure ) 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 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 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 Viability of inoculants: Inoculum quality is a major constraint on germination. Commercial inoculants are often non‐viable (Vosátka et al., 2012 2013 Colonization 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. Dosage: Without sufficient inoculum density (i.e. propagule pressure), colonization will either fail or be functionally ineffective (Allen & Allen, 2005 2015 Order of arrival: There is increasing evidence that priority effects are important for AMF community assembly (Davison 2015; Mummey et al., 2009 2015 1984 1979 2003 2007 2008 2015 Niche requirements: Much has been written on the effect of soil chemistry on AMF, including differential pH tolerance (Porter, Robson, & Abbott, 1987 2010 1993 2010 2005 Persistence 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 2008 2014 2014 2013 2013 2012 1984 2013 1983 2014 2015 1983 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 1994 2005 2011 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. Genetic diversity: AMF used in commercial inoculants may have gone through a bottleneck and have substantially less genetic diversity than natural AMF communities. Spread 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. 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 2014 2016 1988

6 UNINTENDED CONSEQUENCES Even if there is little evidence supporting the efficacy of AMF inoculants under field conditions, there is no evidence about the risk of inoculation to ecosystems (Rodriguez & Sanders, 2015; Schwartz et al., 2006). Inoculation with exotic AMF may have consequences for natural systems, particularly for fungal and plant communities. 6.1 AMF biodiversity One of the few consistent lines of evidence is that inoculation can change AMF communities, leading to partial (Koch et al., 2011; Symanczik et al., 2015) or complete replacement of resident fungi (Jin, Germida, & Walley, 2013; Pellegrino et al., 2012; Symanczik et al., 2015). Not surprisingly, the degree to which an inoculant dominates depends on the context. For example, AMF with r‐life‐history strategies inoculated into communities of K‐strategists could displace resident fungi (Abbott et al., 1983) particularly in disturbed (Antunes et al., 2009) or stressful conditions (Symanczik et al., 2015). Abiotic factors can also influence inoculant dominance—inoculants may fail to establish effective levels of colonization in soils to which they are not well adapted (Abbott et al., 1983). As with establishment, there is a lack of data on long‐term changes to AMF communities following inoculation. As it stands, existing research indicates that the degree to which an inoculant AMF alters resident communities may be difficult to predict. 6.2 Plant biodiversity Inoculation studies that use plant diversity as a response variable overwhelmingly show that inoculation changes plant communities—which may be a desired outcome of inoculation (i.e. for landscape restoration), or an unintended consequence. AMF inoculants have been shown to reduce plant biodiversity and/or prevent the establishment of native plants (Emam, 2016; Koziol, Bever, & Hawkes, 2015; Middleton et al., 2015; Torrez, Ceulemans, Mergeay, de Meester, & Honnay, 2016). Interestingly, inoculant provenance has been shown to be an important predictor of plant community response. For example, native, rather than exotic inoculant AMF may help local plants recover from herbivory (Middleton et al., 2015) or withstand plant invasions (Burkle & Belote, 2015). In contrast, non‐native AMF in commercial inoculants may lead to unwanted promotion of exotic over native plant species.

7 THE ELEPHANT IN THE ROOM: ISN'T EVERYTHING EVERYWHERE? A frequent response to the potential risk of inoculation is that AMF are globally distributed, so inoculation should be of little ecological consequence (Rodriguez & Sanders, 2015). But we know little about the local and global distribution of AMF genotypes and we are only beginning to understand the biogeography and evolutionary biology of the Glomeromycota (Davison et al., 2015; Öpik, Metsis, Daniell, Zobel, & Moora, 2009; Powell & Bennett, 2015). In particular, almost nothing is known about population‐level dynamics in AMF communities (Angelard, Colard, Niculita‐Hirzel, Croll, & Sanders, 2010; Koyama et al., 2017; Rodriguez & Sanders, 2015), but analysis of coding genes indicate that each isolate's genome is unique (Kamel, Keller‐Pearson, Roux, & Ané, 2016). As such, inoculation with a novel genotype may lead to population‐level changes. If these changes result in functional changes to AMF communities (i.e. changes to soil aggregation or to changes in nutrient uptake), then ecosystem functioning may also be affected (see Antunes & Koyama, 2017). Such gaps in knowledge of AMF population and community ecology mean that a predictive framework for the fate of inoculants is a long way off (see Box 1: What drives inoculant spread?)

8 CONCLUSIONS Emphatically, most systems already contain functional AMF communities. Degraded systems may contain highly competitive AMF that have proven tolerance to local conditions, which may be particularly resistant to competition from novel isolates. There is as yet no evidence of “superior” fungal mutualists in the AMF symbiosis, and given the extreme context specificity of the AM symbiosis, it is unlikely that such fungi exist, although there are differences in fungal efficacy. 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 establish at a functional level, or at all, because soil nutrients are too high. Inoculant users may not be able to limit their inoculants to their 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. 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: Horticulture: Closed systems, using artificial soil or hydroponics could benefit from AMF inoculum, since most horticultural crops are highly mycorrhizal. Additionally, as glasshouse agriculture is a closed system, inoculants will not present an environment risk. For horticultural systems where plants are normally transplanted into the field, natural inoculum from local soils would be preferred to commercial inoculants. Severely degraded soils: In some landscape restoration events, inoculum potential is absent (i.e. pit mines, degraded agricultural soil). While practitioners could wait for natural AMF dispersal, in reality, successful restoration of AM systems requires AM plants to establish before non‐AM plants. In this case, AMF inoculation may be necessary to help desired AM plants out‐compete non‐mycorrhizal, ruderal plants. Again, research strongly supports the use of local AMF inocula to negate risks from alien introductions. There remains much to learn about the biology and ecology of AMF before we can adequately develop and control the use of AMF biofertlizers (Figure 3 ). Until then, we suggest that there are suitable conditions that warrant AMF inoculation: Figure 3 Open in figure viewer PowerPoint Knowledge gaps and future research priorities for arbuscular mycorrhizal fungi (AMF) inoculants Given the lack of available data upon which to form best practises, we suggest growers approach the use of AMF inocula conservatively, and only when necessary (Figure 4). Figure 4 Open in figure viewer PowerPoint 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

ACKNOWLEDGMENTS M.M.H. was supported by a Killam Faculty Award, and a Gledden Fellowship. Antreas Pogiatzis, Brittany Altwasser and Negin Kazemian collected data and created maps for Figure 1.

AUTHORS' CONTRIBUTIONS M.M.H. conceived the idea and collected data. M.M.H., P.M.A., L.K.A. (Cambridge, MA) led the writing of the manuscript. M.M.H., P.M.A., L.K.A. and V.B.C. contributed to writing the manuscript. V.B.C. developed charts, figures and developed theoretical models.

DATA ACCESSIBILITY All data are available as Supporting information in the online version of the paper.

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