Plant beneficiating from arbuscular mycorrhizal symbiosis: impact of fungal diversity and soil fertilization

in the presence of bacteria (Hildebrandt et al. 2002). Use of plant roots transformed by Agrobacterium rhizogenes (Bécard & Fortin 1988; Declerck et al. 1996) is the only method to date allowing AMF to undergo their whole life cycle leading to spore production, in an axenic environment. However, this method is not suitable for most of AMF, and is mainly limited to the Rhizophagus and Claroideoglomus genus. Mycorrhizal root colonization can start from a propagule, a broad term that can encompass a spore, a colonized root fragment, an intra-radical vesicle or an extra-radical mycelium (ERM). The type of the propagule able to colonize a plant root varies from one group of AMF to another one (Requena et al. 1996; Klironomos & Hart 2002; Voets et al. 2009). AMF belonging to the Glomeraceae family can colonize a plant root from the different forms of propagule described above, whereas the Gigasporaceae preferentially colonize from the germinating spore (Smith & Read 2008). Although no sexual stages have been reported, the presence of genes involved in mating type have been reported (see 2.1) and some authors speculate on a possible, but not yet observed, sexual phase in the Glomeromycota life cycle (Corradi & Brachmann 2017). As AMF are obligate symbionts, their development has mainly been studied when interacting with the plant. The cell cycle of the AMF is still unknown poorly understood as well as the number of chromosomes (Hijri et al. 2007). Spores, which can be aggregated into groups forming sporocarps, are the only plant-independent fungal structures and can serve as resting structure of the AMF (Smith & Read 2008). The Glomeromycota phylum harbors various genus, which differs in the mode of spore production. Gigasporaceae produce their spores outside of the root and have a more extended extra-radical mycelium, as compared to Rhizophagus, which are producing spores inside the root (Smith & Read 2008). Spore production is very dependent on the plant development. As an example a stress, reducing the C flux from the plant to the AMF, can induce fungal sporulation. Tillage and nitrogen (N) fertilization is also deleterious to AMF spore production (Verzeaux et al. 2017), as ERM can also be an efficient source of propagule, even in field condition. ERM is mainly divided in runner hyphae and absorptive hyphae, with only the runner hyphae having the capacity to colonize a new root fragment (Friese & Allen 1991). Dodd and collaborators (2000) showed that this form of propagule was representative of the Glomus ERM and this feature could not be generalized. In undisturbed soil with native mycorrhizal vegetation, hyphal network is more important than spores or colonized root fragments as inoculum provider (McGee et al. 1997; Kabir 2005). An ERM network with runner hypha is more likely to come in contact with a plant root, than a germinating spore of colonized root fragment (Jasper et al. 2002). The greater colonization efficiency starting from intact ERM is well documented from pot- (Jasper, Abbott, & Robson 1989b; Fairchild & Miller 1990) to field-studies (Kabir et al. 1998; McGonigle et al. 1999; Galvez et al. 2001; Castillo et al. 2006). ERM remained also infective under field conditions (Kabir et al. 1997), with temperature falling to -2°C, or when air temperature varied close to 30 °C for almost 4 month with no rain fall or no surface vegetation (Brito et al. 2011). So, although AMF survive at low temperature, their ability to colonize plant roots does not require any specific temperature shift.

AMF are present in the whole ecosystem with no geographical structuration, with identical genotypes present on different continents (Rosendahl et al. 2009). Rosendahl and collaborators (2009) found a recent diversification of Glomus mosseae (recently renamed in Funneliformis mosseae), suggesting that the speciation and the expansion happened after the continental spread. Lack of endemism results have also been found by Davison and collaborators (2015) even though species concept and actual limitations for AMF identification (see below) could be misleading as the defined species could encompass several local endemic species (Rosendahl et al. 2009; Bruns & Taylor 2016). As the species definition in the AMF community remains an open question (Bruns et al. 2017), dispersal of AMF is also unclear. Therefore, with available molecular tools to track AMF, the question of their vicariance is still an open question. However, F. mosseae is preferentially detected in agricultural fields, suggesting that human activities could be an important driver for the selection and/or dissemination of AMF (Allen et al. 1987; Allen 1991; Rosendahl et al. 2009). An anthropogenic trait could also be applied to the AMF isolate Paraglomus sp HMCl5, molecularly detected in only two locations, a zinc mining site in Poland (Turnau et al. 2001) and an orphan zinc mining site in south of France (Sánchez-Castro et al. 2017). Polish, miners having worked in both mining sites, they could have contributed to the dissemination of this fungus well adapted to these extreme environmental conditions.
Plant starvation is an important signal for the AMF to sporulate, as spore allows its survival in the absence of plants.

II. Is there information about the relationship of mycorrhizal diversity and health status of soil and subsequent the health status of the corresponding plant species?

2.1 Mycorrhiza species concept
Arbuscular mycorrhiza are the most common form of mycorrhiza on the planet, and are present since the Devonian (Pirozynski & Dalpe 1989; Taylor et al. 1995; Redecker et al. 2000) when the land plant colonization of the Earth started. So, for more than 450 M years, arbuscular mycorrhiza fungi (AMF) and plants have been in interaction and co-evolved. As the American plant pathologist, Stephen Wilhelm, said: “…in agricultural field conditions, plants do not, strictly speaking, have roots, they have mycorrhizas”. This provocative statement reflects a widespread situation often overlooked, partially because of the difficulties to assess a functional mycorrhizal interaction. AMF are ubiquitous and form a symbiotic interaction with more than 90% of land plant families (Wang & Qiu 2006) and are not strictly speaking host specific. However, numerous works have shown that plant and AMF diversity are linked (van der Heijden et al. 1998; Hartnett & Wilson 1999; Klironomos et al. 2000; Bever et al. 2001; Xiang et al. 2014) and that soil use and management affects AMF diversity (Gollotte et al. 2004; Brito et al. 2008; Xiang et al. 2014; Moora et al. 2014; i.e. Ciccolini et al. 2016).
Analysis of the AMF diversity is closely dependent on the detection method (Taylor et al. 2006). Two main approaches have been used. The first approach was to estimate the AMF diversity through the morphological species recognition (MSR) of fungal spores isolated from the field (Oehl et al. 2003; i.e. Oehl et al. 2017) or from trap culture (i.e. Mathimaran et al. 2007; Leal et al. 2009). The MSR method allows only the identification of the AMF sporulating at the sampling time; a direct link to the AMF species colonizing the plant root is difficult when various plants are present and some of the AMF isolates colonizing the plant roots may not be sporulating at sampling time. Moreover, the MSR method is based on the morphological identification of an asexual spore. The small size of this organ when used for the species identification, reduces the number of evolutionary relevant characters (Taylor et al. 2006), leading to a reduced number of AMF morphologically identified species (Redecker et al. 2013). The morphological identification is also not trivial; isolates belonging to the same species on morphological basis, can however differ significantly in some of their biologically important traits (Munkvold et al. 2004), such as the extra-radical mycelium (ERM) length, a trait not taxonomically relevant. To overcome these challenges, the second strategy, the phylogenetic species recognition (PSR), using molecular methods to assess the AMF diversity starting from roots or directly from soil DNA extracts, was developed (i.e. van Tuinen et al. 1998; Husband et al. 2002; Gollotte et al. 2004; Long et al. 2010; Öpik et al. 2013; Stockinger et al. 2014; Wei et al. 2015; Bouffaud et al. 2016; Bouffaud et al. 2017). PSR approaches have been widely used, and were recently improved in depth with the development of sequencing technologies (Öpik et al. 2013; Hart et al. 2015). However, these approaches have also some limitations. For example, it is well known that the ribosomal operon of AMF are polymorphic, even within the same nucleus. This implies that different ribosomal sequences can be obtained from the same spore requiring the use of an arbitrary threshold to group the ribosomal sequences in a so called Virtual Taxa or Operational Taxonomic Unit (Öpik et al. 2010). The arbitrary threshold generally used for PSR of AMF is of 97%. It has to be kept in mind that the small ribosomal sub-unit (SSU) region of human matches at 99% to that of house mouse (Mus musculus) (Bruns et al. 2017), and that AMF isolates of the same species can differ significantly in some properties not taken into account by MSR (Munkvold et al. 2004). Therefore, the link between ribosomal sequences and AMF species is not trivial and is dependent on the taxonomic concept of species (Bruns et al. 2017). For example, Davison and collaborators (2015) estimated on the basis of the SSU diversity, that AMF showed limited endemism at the world scale level. This point of view was contested by Bruns and Taylor (2016) who pointed out the reduced level of discrimination inherent in the SSU. The consequence of the use of the SSU to establish the so-called Virtual Taxa could be that \”species\” in the Glomeromycota could actually be a collection of fairly distant related taxa. Virtual Taxa is an interesting and practical approach, similar to Operational Taxonomic Units (OTUs), to assess the AMF diversity in contrasting environments (Öpik et al. 2010), but can be misleading as a taxon, is the taxonomic unit and should be independent of any arbitrary unit or threshold. When establishing taxa, on the basis of a single molecular sequence, one has to be very cautious. It has been shown in yeast that a correct phylogenetic tree is obtained with at least 7 reference genes (Rokas et al. 2003). Taylor and collaborators (2006), discussed the observation that the inferred geographic range of an AMF species depends on the method of species recognition. Fifteen distinct haplotypes were found between 17 isolates of R. clarus, although their large ribosomal sub-unit (LSU) differed by less than 2% (Purin & Morton 2013).
A third strategy to assess AMF diversity and to establish fungal species would be the use of anastomosis, a biological species recognition (BSR) method. AMF lack obvious sexual structures even if about 85% of the core meiotic genes (i.e. HOPP2: homologous-pairing protein 2, a MND1: meiotic nuclear division protein), and mating-type gene homologues and putative sex pheromone-sensing mitogen-activated protein (MAP) kinases are present in the genome of Rhizophagus irregularis DAOM197198 (Halary et al. 2011; Tisserant et al. 2012; Halary et al. 2013; Corradi & Brachmann 2017). But, as the function of the proteins coded by these genes is unknown, it is not yet possible to conclude that a cryptic sexuality could occur in AMF (Corradi & Bonfante 2012). AMF hyphae are coenocytic and produce asexual spores with multiple nuclei, and hyphae originating from closely related spores can perform self-anastomosing (Giovannetti et al. 2003). About 95 % of fusions can occur within the same hypha by a mechanism known as wound healing (La Providencia et al. 2005; Voets et al. 2006). The development of anastomosis between AMF isolates depends either on their vegetative compatibility or on their geographic origin (Giovannetti et al. 2003; Purin & Morton 2013), with most anastomosis events occurring between hyphae originating from the same spore (Giovannetti et al. 1999). Although this BSR method could be an alternative to the PSR method, the incapacity to grow AMF in the absence of a host plant, reduces the feasibility to use this approach for diversity analysis.
AMF are supporting a wide range of ecosystemic functions such as increased plant tolerance to biotic or abiotic stress, increased soil stability, secondary metabolism or promotion of plant growth (for revue see Gianinazzi et al. 2010). The latter, being easy to assess, has often been used as indicator for a beneficial effect of AMF diversity on plant growth. Studies on complex mycorrhizal communities (i.e. van der Heijden et al. 1998; O\’Connors et al. 2002; Wagg et al. 2011) showed that plant growth was positively impacted by AMF diversity, and studies on more simplified systems showed that this growth response was very dependent both on the AMF and the plant (i.e. Klironomos et al. 2000; Cavagnaro et al. 2005; Gosling et al. 2016). Klironomos and collaborators (2000) showed that Gigaspora margarita had a beneficial effect on the biomass of Agrosistis gigantea but an inhibitory effect on growth of Daucas carota, suggesting a continuum from symbiosis to parasitism when plant biomass is used as proxy for the beneficial effect of the mycorrhizal symbiosis. More recently, Courty and collaborators (2015) compared leek and sorghum which are respectively a C3 and a C4 plant. They showed that the nutrient exchanges between the plant and the fungus were dependent on the plant/fungal couple, suggesting a high variability in the plant responsiveness to a mycorrhizal colonization. A mycorrhizal colonization can therefore be beneficial for one function but not for another. The beneficial impact of a mycorrhizal symbiosis is not directly related to the level of root colonization, but to a certain extend to the development of the ERM (Sawers et al. 2016) that is highly variable even within the same species (Munkvold et al. 2004)

2.2 Common mycorrhizal networks in sustainable agriculture
What’s that? AMF have nearly unrestricted host ranges and can associate with the majority of plants species (Smith & Read 2008). Perennial plant species harbored a lower AMF diversity than annual plant species, and half of the AMF species that were identified were specific for one plant species (Torrecillas et al. 2012) suggesting that the establishment of designed AMF communities in agricultural applications for enhanced crop productivity is not trivial. AMF are forming ERM networks spreading from colonized roots into the surrounding soil (Giovannetti et al. 2015) and extending from 2.7 to 30 m per gram of soil, depending on the fungal species (Giovannetti et al. 2003; Mikkelsen et al. 2008). Length of intact ERM depends on the fungal species and the associated plant species: the ERM can range from 5 to 7.4 m for F. mosseae in association with Thymus vulgaris and Alium porum respectively (Giovannetti et al. 2015). The mean growth could range from 3.1 mm to 3.8 mm per day for F. mosseae and F. caledonius in association with Trifolium subterraneum mycorrhizal roots (Giovannetti et al. 2001; Mikkelsen et al. 2008), and to 738 mm per day for F. mosseae in association with T. vulgaris (Giovannetti et al. 2015). The ERM of one AMF or hyphal amalgamation/fusion of separated mycelia (Giovannetti et al. 2004; Selosse et al. 2006; Mikkelsen et al. 2008) can colonize and further connect neighboring plants of same or different species in a community to form common mycorrhizal networks (CMNs) (Barto et al. 2012). CMNs benefit host plants in many ways and transfer may be bidirectional between plants, with a net flux toward one plant (Selosse et al. 2006; Simard et al. 2012). CMN may improve seedling establishment (van der Heijden 2004), influence plant and microorganism community composition (van der Heijden & Horton 2009), induce an efficient nutrient exchange, improve interplant nutrition (He et al. 2013) and growth through plant-plant facilitation (Zhang & Franken 2014). Moreover, CMN may induce plant defense response (defensive enzyme activities and defense-related gene expression) and plant communication through for example a variety of phytohormones as jasmonic acid, methyl jasmonate and zeatin riboside (Song et al. 2010).

2.2.1 Plant-plant competition. CMNs have been found to amplify intraspecific competition by altering its population size-class distribution (Weremijewicz et al. 2017) a functional trait reflecting either a symmetric or an asymmetric competition (Weiner 1985). Generally, the distribution of the populations is symmetrical shortly after germination and evolves towards an asymmetrical distribution with the age of the plants, reflecting dominance of large individuals getting a disproportionate share of a limiting resource (Weiner & Thomas 1986). CMN play a role in plant root competition and by extension on mineral nutrient acquisition: plants with intact CMN showed an asymmetric competition whereas plants with severed CMN showed a symmetric competition (Weremijewicz & Seto 2016), suggesting that intact CMN may supply nutrients as nitrogen to large individuals that are highly photosynthetically active and that provide the most carbon to their associated AMF (Merrild et al. 2013; Weremijewicz et al. 2016). This reciprocal reward could be dependent of the rate of exchange of mineral nutrients for carbon from host plants (Kiers et al. 2011). Other factors may influence the dynamics of nutrients in CMN as within-species size hierarchies and between-species interactions (Weremijewicz et al. 2017), and host sink strength (Walder & van der Heijden 2015). However, the reciprocal reward seems not being a general case as shown by Walder and collaborators (2012) for a CMN built between sorghum and flax. This indicates that resource exchange in the AM symbiosis is controlled by biological market dynamics (Walder & van der Heijden 2015)., and there are indications that the cost to nutrient benefit ratio varies among different host plant species (Walder et al. 2012).
Effects of CMN on seedling recruitment in contrast to the waiting of AMF spore germination may be beneficial. AMF spore germination may represent more a cost rather than a benefit for the developing seedling as carbon requirements is higher for Gigasporaceae compared to Glomus species (Thomson et al. 1990; Feddermann et al. 2010; Lendenmann et al. 2011) for the development of the ERM (Chagnon et al. 2013). Moreover, phosphorus resources of the seedlings may be limited (Koide 1985; Ronsheim 2012) and the net outcome of the relationship for the plant with different AMF species is variable and dependent on the benefit analyzed (Johnson et al. 1997; Hoeksema et al. 2010; Kiers et al. 2011; Smith & Smith 2013). In contrast, effects of CMN on plant germination (growth and chances for establishment) are positive when seedlings get tapped into the existing CMN (van der Heijden 2004). CMN may provide a faster mycorrhizal formation, limit the investment of the seedling in the construction costs of the hyphal network and give access to nutrients and water and possibly even carbon derived from other plants connected to the CMN (Varga et al. 2017).

2.2.2 Plant-CMN-plant communications and potential impact on crop pest control. Plant-plant signaling could help achieve food security by minimising crop losses due to pests. The potential role of AMF on competition through allelopathy has been noted (Barto et al. 2011; Achatz et al. 2014). The CMN can act rapidly (from 24h to 50h) as signaling conduits for signaling compounds (Babikova, et al. 2013; Johnson & Gilbert 2015) after necrotrophic fungal attack (Song et al. 2010) (Song et al. 2010) and caterpillar attack (Song et al. 2014). The CMN helps in extending the bioactive zone of allelochemicals in the soil (Sanon et al. 2009; Barto et al. 2012; Hale & Kalisz 2012) or in changing the volatile organic chemicals from their leaves (Babikova et al. 2013). So, CMNs constitute a considerable potential for crop pest control through this underground plant-plant signaling mechanism (Babikova et al. 2014). The reliability whether this is rapid enough for realistic crop pest control, will depend on (i) the rapidity and frequency of enemy attack, (ii) the number of attacked crop plants, (iii) the signal travelling over long distances (at least 20 cm on plant beans; (Babikova et al. 2013; Babikova et al. 2013) (iv) the putative relay of the signals between plants and (v) the putative transfer to other CMNs. However, the most fundamental requirement for CMN for being efficient and useful in crop pest control is to remain warn of enemy attack and physically intact whereas most cropped soils are tilled and this is likely to break up the CMN (Brígido et al. 2017). A mycorrhizal symbiosis has to be fully established to be active (Slezack et al. 2000), achieved more rapidly when the fungal inoculum source is an intact ERM, as compared to mycelium or mycorrhized root fragments (Brígido et al. 2017). This is also observed in field experiments, where it has been observed that mycorrhizal plants colonisation decreases with increasing tillage intensity (Carpenter-Boggs et al. 2003) and tillage could provide selective pressure for fungal species that are more tillage-tolerant, which may change the community composition of fungi (Brito et al. 2012) and might impact on their ability to transfer defense signals.

2.3 Mycorrhizal growth benefits. In a mycorrhizal plant, the majority of the P uptake is achieved through the AMF pathway regardless of the importance and direction of the mycorrhizal response and the total P uptake (Smith 2003; Smith et al. 2004). AMF are not equally beneficial for the host (Johnson et al. 1997; Munkvold et al. 2004; Smith et al. 2009; Johnson & Graham 2013), beneficial effect which can vary from one species to another (Gosling et al. 2016). The beneficial effect of AMF to the plant is not limited to the phosphate nutrition (Gianinazzi et al. 2010), although it is one of the most studied mycorrhizal effect, and more functions such as: increased tolerance to biotic or abiotic stress, improvement crop nutritional quality (Farmer et al. 2007), improvement soil stability (Burrows 2014), should be taken into account to have the most objective picture of the mycorrhizal impact on plant fitness. In natural ecosystems, plants could be colonized by dozens of species with apparently no direct link between mycorrhizal growth benefit and AMF phylogeny (Novais et al., 2014; Mensah et al. 2015; Koch et al., 2017), and we could hypothesize that the high genetic variability among different isolates could derive from a co-evolution between co-existing fungal and plant populations.

2.4 Mycorrhizal diversity and plant health status.
Taking into account the sub mentioned considerations, plant general health status is very dependent on the development of the mycorrhiza interaction. Plant growth response of mycorrhizal plants is substantially lower when colonized by one fungal partner compared to plants colonized by multiple fungal species (Hoeksema et al. 2010), and plant response to multiple AMF depend on mutualistic quality (Kiers & Denison 2008), and several models have been proposed :
– positive mutualistic association (i.e. Walder et al. 2012) if (i) the functional effects of the different members of the AMF community are complementary (Hart & Reader 2002), (ii) closely related AMF coexist in the AM community, (iii) more beneficial and less “pathogenic” AMF community establish a symbiosis and (iv) the biological market dynamics is in favour of the host (Bücking et al. 2016).
– negative mutualistic association (Violi et al. 2007; Gosling et al. 2016) if the functional traits of AMF are more redundant than complementary leading to antagonistic effects and competition rather than synergetic effects (Maherali & Klironomos 2007; Jansa et al. 2008), however the despite the negative effect of interspecific interaction on plant.
The mycorrhizal effect can be very variable depending the mycorrhizal fungus interacting with the plant, as well as the ecosystemic services observed (Farmer et al. 2007). Environmental conditions for the host aboveground play also a role in the competitive interactions among fungi belowground (Knegt et al. 2016). But a factor which is constant in the mycorrhizal interaction, mycorrhization at the early steps of the plant development is to most beneficial (Farmer et al. 2007; Brígido et al. 2017), and the integrity of the ERM is a key factor for an optimum expression of the mycorrhizal promotion of the plant development (Walder et al. 2016; Brígido et al. 2017).
III. Is there information about the rejection of mutualism by corresponding plant?
Mutualism consists of cooperative interactions between members of different species that benefits each other (Johnson et al. 1997) and often involves the trade of resources (Wyatt et al. 2016). In many environments, plant growth is limited by inadequate nutrient supply, a situation that can be alleviated by symbiosis with soil-borne AMF of the sub-phylum Glomeromycotina (Spatafora et al. 2017). In this interaction, plant-produced C is exchanged against AMF-acquired soil mineral nutrients, mainly P and N. This is achieved by the fungal ERM emanating from the root system, which reaches far beyond the rhizosphere and therefore can acquire nutrients from soil volumes to which roots have no access (Friese & Allen 1991). The nutrients that are taken up by the fungus are then transferred to the intra-radical mycelium in the root cortex, and subsequently to the host through the development of highly branched fungal structures called arbuscules. Consequently, in mycorrhizal plants, two pathways can mediate nutrient acquisition from the soil solution: the direct plant uptake pathway at the root–soil interface, and the mycorrhizal pathway that extends from extra-radical hyphae to arbuscules (Gutjahr & Parniske 2013).
Since AMF are obligate biotrophs, that cannot grow apart from their hosts as they are completely dependent on delivery of photosynthetically fixed C, the C supply from the plant is regarded as an obvious benefit for the symbiont (Konvalinková & Jansa 2016). Whether plant growth profits from being mycorrhizal is usually viewed as a question of C cost versus benefits through improvement of nutrient uptake by AMF (Corkidi et al. 2002). For plants, mutualism in terms of positive mycorrhizal growth response is notably expected when nutritional benefits to the plant exceed plant costs for fungal sustenance (Johnson et al. 2015; Friede et al. 2016). When plants are nutrient-limited, they profit from allocating carbohydrates to the fungi, as, in turn, the symbionts provide them with immobile soil mineral nutrients (Johnson et al. 1997). When all required elements are available without mycorrhiza, the C cost for fungal sustenance exceeds the benefit and plants can stop allocating C to the symbionts to save energy (Blanke et al 2005a). Consequently, AMF become C-limited and mycorrhizal colonization can be inhibited (Treseder & Allen 2002).
As detailed below, plants have long been known to respond to high P levels with suppression of symbiosis. In addition, recent studies have demonstrated an interplay between P and N availabilities to control AM associations (Bonneau et al. 2013). It has then been suggested that the plant can assess not only its own costs but also the benefits provided to the fungal partner (Nouri et al. 2014). With regard to the mechanisms involved in suppression of symbiosis by the host plant, most current knowledge deals with impaired carbon delivery to the fungus in the forms of sugar and lipid compounds.

3.1. Is plant control of AM colonization dependent upon phosphate availability?
P is considered as the second most important plant nutrient after N that is critical for plant growth (Balemi & Negisho 2012). As P is often present in unavailable forms or in forms that are only available outside of the rhizosphere, the application of soluble orthophosphate ions (H2PO4- and HPO4-2), the inorganic phosphate (Pi) forms taken-up by the roots, is necessary to ensure plant productivity in most agricultural systems (Schachtman et al. 1998; Smith & Smith 2011). Plants, however, have long been known to respond to high soil Pi levels in the range of 100 mg or more of Pi per kg of soil (Schubert & Hayman 1986; Baon et al. 1992; Corkidi et al. 2002), with suppression of symbiosis (Baylis 1967; Mosse 1973; Sanders & Tinker 1973). This phenomenon is referred to as Pi inhibition of mycorrhizal colonization (Graham et al. 1981). The adverse effect of high soil P levels on AM formation is primarily due to high P concentrations in the roots (Sanders 1975), which results in reduced C allocation to the AMF after several weeks of symbiotic functioning (Olsson et al. 2002; Olsson et al. 2006). Although the inhibitory effect of high Pi level on C transfer is true on the long term, high Pi has a stimulatory effect on the short term (Olsson et al. 2006). This indicates that roots maintain C transfer to the fungus until they reach a satisfactory intracellular Pi status and do not directly respond to the external Pi level (Javot et al. 2007).
The diagnostic feature of AM symbiosis is the arbuscule, a specialized tree-like structure enveloped by an extension of the host plasma membrane, the peri-arbuscular membrane that separates the fungus from the plant cell cytoplasm. This delineates the interface compartment, an apoplastic space that surrounds the fungus and mediates plant Pi uptake (Fitter 2006; Bonfante & Genre 2008; Gutjahr & Parniske 2013). Upon high Pi supply, many studies have highlighted that fungal structures show a significant reduced number of arbuscules (Rausch et al. 2001; Breuillin et al. 2010; Bonneau et al. 2013; Kobae et al. 2016)1. When exploring the transcriptional changes associated with elevated Pi supply in inoculated Petunia hybrida, Breuillin et al. (2010) noticed that repression of AM-inducible Pi transporter (PT)-encoding genes upon Pi addition preceded the reduction in colonization, indicating that PT loss-of-function actually represents a cause, but not a consequence, of apparent decreased symbiosis. Currently, AM-inducible Pi transporters, which all belong to the clade Pht1, have been identified in many plant species (Javot et al. 2007). Pi transporters belonging to subfamily I such as OsPT11 of rice and MtPT4 of Medicago truncatula, are only induced during AM symbiosis and their location in the arbuscule-branch domain of the peri-arbuscular membrane, has been demonstrated (Pumplin & Harrison 2009; Kobae & Hata 2010). Consequently, they are referred to as mycorrhiza-specific transporters (Javot et al. 2007). Importantly, reduced fungal growth in planta and altered arbuscule morphology in Medicago pt4 and rice pt11 mutants revealed that PT4/PT11 is not only essential for AM-mediated phosphate uptake but also for arbuscule maintenance (Javot et al. 2007; Yang et al. 2012). In addition, P treatment interrupts the development of arbuscules at the coarse hyphal branch stage, suggesting that the transcriptional regulation of genes involved in arbuscule branching may be suppressed during Pi inhibition (Park et al. 2015; Kobae et al. 2016). This indicates that plants allow continuation of arbuscular development and mycorrhizal fungal existence in roots only when soil P captured by the hyphae is transported into the roots (Javot et al. 2007). These finding support the model proposed by Fitter (2006) according to which a successful fungus must continue to provide the plant with Pi through the arbuscules in order to maintain a reciprocal C flux. Consistently, it has been shown that the host is able to reward the symbiont with carbon for efficient symbiotic phosphate delivery (Bever et al. 2009; Kiers et al. 2011). Taken together, current available data suggest that Pi delivered to the root cortical cell could trigger a signal that controls carbon release to the fungus (Javot et al. 2007; Breuillin et al. 2010; Gutjahr & Parniske 2013; Wang & Wu 2017).

3.2. Is plant control of AM colonization dependent upon inorganic phosphate and nitrogen availabilities?
N is the nutrient whose availability most commonly limits plant growth in natural ecosystems (Balemi & Negisho 2012; Vitousek & Howarth). Many studies have established that arbuscular mycorrhizal fungi transfer inorganic N to the host plant (reviewed by Corrêa et al. 2015; Bücking & Kafle 2015; Chen et al. 2017). AMF can take up nitrates (NO3−) and ammonium (NH4+) from the soil (Hodge et al. 2010; Bücking & Kafle 2015), and similar to Pi, the AM uptake pathway can also contribute significantly to N nutrition in the host plant (Tanaka & Yano 2005). It has been estimated that close to one-third of the N in root protein amino acids can be provided by symbiotic AMF (Govindarajulu et al. 2005). Some aspects of inorganic N and P transfer in the symbiosis are very similar. Following uptake in the extraradical hyphae, they are translocated to the intraradical hyphae via the vacuole in a storage form (poly-P or arginine), and the ionic forms (Pi or NH4+) are released to the plant apoplast, and subsequently taken up by mycorrhiza-inducible transporters (Javot et al. 2007). Notably, in Glycine max, Sorghum bicolor and M. truncatula, the ammonium transporters AMT4;1, AMT3;1 and ATM2;3 have been localized on the branch domains of the peri-arbuscular membrane, indicating that active ammonium transfer occurs around the arbuscule branches (Kobae & Hata 2010; Koegel et al. 2013; Breuillin-Sessoms et al. 2015). In Lotus japonicus, LjAMT2;2 absorbs N released by AMF in the symbiotic interface and is required for mycorrhizal symbiosis. Unlike other plant AMTs, LjAMT2;2 transports NH3 instead of NH4+, indicating that, in addition to NH4+, NH3 might be released at the symbiotic interface by AMF and taken up by plant hosts (Guether et al. 2009; Bücking & Kafle 2015).
As in the case of Pi fertilization, inorganic N fertilization in the range of 100 mg or more of N per kg of soil, also has been shown to reduce root colonization by AMF (Lanowska 1966; Chambers et al. 1980; Azcón et al. 1982; Corkidi et al. 2002; Blanke, Renker, Wagner, Füllner, Held, Kuhn, & Buscot 2005b). Additionally, high external N level around mycorrhizal roots can reduce the C allocation to the fungus (Olsson et al. 2005). The response of mycorrhiza to fertilization, however, is highly context dependent. Johnson and collaborators (2003; Johnson et al. 2015) found that N addition negatively affected AM colonization of roots in soils with low N:P ratios and positively affected AM colonization in those with high N:P ratios. According to the law of the minimum stating that plant production may be controlled by a single essential resource that is in limiting supply (Liebig 1843; van der Ploeg et al. 1999), it has therefore been proposed that the relative availability of soil N and P determines whether or not mycorrhizal benefits out-weigh their costs (Johnson 2010; Johnson et al. 2015). This trade-off balance model notably predicts that N fertilization is only beneficial if the plant is limited by P and will therefore benefit from providing C to the roots and mycorrhizal fungi. By manipulating nutrient and light availabilities, experimental evidence shows that inorganic N sources can indeed elicit a mutualism scenario predicted by the trade-off balance model, whereby both plant and fungi benefit from the addition of a rich N source in a P limited system (Johnson et al. 2015). Additional evidence that this response is driven by the C-to-nutrient exchange dynamics was provided by Fellbaum and collaborators (2012).
In agreement with the Liebig\’s law of the minimum, long-term P inhibition of AM symbiosis is partially suppressed under low N conditions, suggesting that plants promote the AM-symbiosis as long as they are limited by one of the two major nutrients (Blanke, Renker, Wagner, Füllner, Held, Kuhn, & Buscot 2005b; Nouri et al. 2014). In support of the idea that arbuscule lifespan is regulated in part by N, premature arbuscule degeneration is relieved when plants are deprived of N (Javot et al. 2011). However, the recovery of AM colonization did not lead to increased N levels in these plants, suggesting that N starvation triggers a signal that promotes AMF colonization (Blanke, Renker, Wagner, Füllner, Held, Kuhn, & Buscot 2005b; Nouri et al. 2014; Bücking & Kafle 2015). Consistently, functioning of the peri-arbuscular ammonium transporter AMT2;3 is required for the low-N suppression of the premature arbuscule degeneration in pt4 mutants, but without significant changes in symbiotic N transport (Breuillin-Sessoms et al. 2015). The authors thereby proposed that transport of Pi or ammonium through the respective symbiotic transporters acts not only to deliver nutrients to the root cell but also initiates an unknown signalling mechanism that enables the maintenance of arbuscules. This raises the question whether the inhibition of AM development by Pi may become attenuated when plants are limited for nutrients other than nitrogen (Nouri et al. 2014). Using petunia plants inoculated with Rhizophagus irregularis, they found that only Pi and nitrate exerted a negative influence on AM root colonization, whereas other plant major nutrients such as potassium, calcium, magnesium, sulphate, and iron did not influence mycorrhizal development at elevated concentrations.

3.3 Potential mechanisms involved in fugal spread limitation through reduced plant carbon allocation
While there is evidence that plants can reduced carbon allocation to the fungus in response to high Pi or N fertilization regime (Olsson et al. 2002; Olsson et al. 2006; Kiers et al. 2011), the mechanisms by which this is achieved are still unclear and essentially documented in Pi-replete plants. With regard to plant C supply in the form of sugars, sucrose (Suc), which represents a substantial portion of the photosynthetic fixed CO2, is used for long-distance carbon and energy transport into diverse heterotrophic sinks and consists of the preferred carbohydrate translocated to the mycorrhizal interface (Bücking & Shachar-Hill 2005). Because intra-radical fungal structures are unable to take-up Suc, the latter is believed to be hydrolysed before fungal utilization by cytosolic Suc synthases (SucS), producing UDP-glucose and fructose, or by invertases (Inv), producing fructose and glucose, which is the main form taken up by the AMF (Solaiman & Saito 1997; Pfeffer et al. 1999). Potential mechanisms involved in limiting fungal spread in planta may thus include down-regulation of SucS and Inv. Notably, inhibition of Inv functioning resulted in reduced apoplastic invertase activities in roots, lower contents of glucose and fructose, together with a diminished mycorrhization (Schaarschmidt et al. 2007). Likewise, silencing of MtSuc1 led to an internal mycorrhization-defective phenotype as inferred from reduced frequencies in internal hyphae, vesicle and arbuscule development (Baier et al. 2010). In addition, according to the transcriptional activation in arbusculated cells of the sugar transporter MtHex1 that can mediate glucose uptake, a re-uptake of apoplastic hexoses by the cognate protein has been proposed as another mechanisms to limit fungal spread in planta (Gaude et al. 2011; Roth & Paszkowski 2017). At least with respect to the transcriptional data relative to sugar metabolism in whole plant petunia roots, Breuillin et al. (2010), however, discarded the idea that high Pi supply may limit AMF intra-radical development by reducing sugar delivery to the symbiont. Nonetheless, post-transcriptional events such as internalization of the sucrose transporter/sensor SUT2 by endocytosis (Bitterlich et al. 2014) also can contribute to contain fungal expansion (Roth & Paszkowski 2017). In addition, AMF have the ability to acquire sugars through their intra- and extra-radical hyphae via the monosaccharide transporter MST2 (Helber et al. 2011). MST2 was found to be able to transport glucose and fructose, but also xylose, mannose, galactose, glucuronic and galacturonic acids that are components of the cell wall-like apoplastic plant-fungus interface. This corroborates the idea that AMF can indeed feed on cell wall components (Smith & Smith 1990). Furthermore, MST2 in planta expression was clearly found to depend on the symbiotic phosphate delivery pathway. Namely, upon Pi fertilization, the expression of MST2 was down-regulated concomitantly with that of the mycorrhiza-specific Pi transporter PT4. Knockdown MST2 lines through host-induced gene silencing indicate that MST2 is indispensable for a functional symbiosis as inferred from lower mycorhization levels coupled to the appearance of not fully developed and early senescing arbuscules, a phenotype that parallels the abolished expression of MtPT4 (Helber et al. 2011). This notably confirms the importance of plant carbohydrates for the maintenance of intra-radical hyphae and arbuscules (Roth & Paszkowski 2017). Consistent with this view, down-regulated genes upon mycorrhization in poplar roots under high-N conditions include genes involved in carbohydrate metabolism, glycolysis, and tricarboxylic acid cycle, indicating that under full nutrient conditions the plant reduces transfer of carbon as it has access to all essential nutrients by itself (Calabrese et al. 2017). In addition to sugars, recent cumulative evidence shows that carbon is also provided from the host plant to the AMF in the form of fatty acids (FAs) (Bravo et al. 2017; Jiang et al. 2017; Luginbuehl et al. 2017; Keymer et al. 2017). As reviewed by McLean et al. (2017), Rich et al. (2017), Wang et al. (2017), Roth and Paszkowski (2017) and Luginbuehl and Oldroyd (2017) (2017), current knowledge indicates that the AM-specific plant thioesterase FatM releases 16:0 FAs (palmitic acid) which when attached to CoA, are used as a substrate by the glycerol-3-phosphate acyl transferase (GPAT) RAM2 to produce 16:0 β-monoacylglycerol. This compound can be exported across the peri-arbuscular membrane by the half ABC transporters STR and STR2. Although the influence of Pi availability on the plant proteins that direct lipid flux within arbuscules yet have not been investigated, the mycorrhiza-specific GPAT belongs to the genes expressed in all mycorrhiza fertilized with low phosphate but not in low or high P control roots (Breuillin et al. 2010). Moreover, it was also noticed that the expression of STR and STR2, which mediate lipid flux to AMF, is also repressed by high Pi levels (Wang et al. 2017). Taken together, these findings suggest that, upon Pi supply, the symbiont may be starved for plant C in the form of lipids.

In summary, available knowledge indicates that plants control the degree of AM colonization in function of their nutrient requirements, according to Liebig\’s law of the minimum. Currently, Pi and N have been identified as the major nutritional determinants of the interaction (Nouri et al. 2014). It is believed that nutrients delivered to the root cortical cell can trigger a signal that controls C release to the fungal partner. The rationale behind this strategy is that a symbiont unable to deliver significant levels of soil nutrients would only have access to low levels of C available in the root apoplast (Javot et al. 2007). While the nature of this signal is unknown, data relative to Pi-replete plants indicate that the plant host may restrict arbuscule development not only by reducing sugar but also lipid delivery to the symbiont. To date, twelve genes coding for enzymes involved in the biosynthesis of plant lipids have been identified through phylogenomics as only required for AM symbiosis (Bravo et al. 2016). Future knowledge regarding their regulation upon high Pi or N fertilization regimes should shed light about the role of plant lipids in the regulation AM symbiosis development.