Trading Carbon Between Arbuscular Mycorrhizal Fungi and Their Hyphae-Associated Microbes

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Trading Carbon Between Arbuscular Mycorrhizal Fungi and Their HyphaeAssociated Microbes B. Drigo1,2, S. Donn1 1Western

Sydney University, Penrith, NSW, Australia; 2University of South Australia, Mawson Lakes, SA, Australia

22.1  MYCORRHIZAS AND HYPHAE-ASSOCIATED MICROBES The most widespread symbiosis in the rhizosphere is the symbiosis between plant roots and mycorrhizal fungi (Smith and Read, 2008). Mycorrhizal fungi affect the exchange of nutrients at the plant root level (Brundrett, 2002), and have impacts on ecosystem functioning through their effects on plant productivity and community assembly (Bever et al., 2010). Mycorrhizas develop mutualistic interactions that allow the plants to exploit habitats that would be otherwise inaccessible to them, and boost their competitiveness over plants lacking these associations (Bever et al., 2010; Smith and Read, 2008). The origin of mycorrhizal symbiosis, based on molecular clock dating, is estimated to have occurred around 600 million years ago (Redecker et al., 2000), supporting the hypothesis that those mycorrhizas were instrumental in the colonization of land by plants (Remy et al., 1994). The most ancient and widespread type of mycorrhiza is the arbuscular mycorrhiza, established by many plant species and a narrow taxonomic group of arbuscular mycorrhizal (AM) fungi (Chapter 1), followed by ericoid, orchid, or ectomycorrhizal (EcM) systems (Meharg and Cairney, 2000; Smith and Read, 2008; Schüßler et al., 2001). Nowadays, mycorrhizal fungi occur in the majority of existing plant families (Smith and Read, 1997) and have a direct influence on biogeochemical cycles and an indirect effect on ecosystem functions (van der Heijden et al., 2008; Veresoglou et al., 2012), structure, and productivity (van der Heijden et al., 1998; Jansa et al., 2008). Most plant roots are colonized by multiple mycorrhizal fungi and most mycorrhizal fungi are not host-specific, colonizing various host plants at the same time (van der Heijden et al., 2015). As a consequence, plants are usually interconnected by mycorrhizal mycelial networks in so-called “woodwide webs” (Simard et al., 1997).

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With the mycorrhizal hyphae on one side connected to plant roots, and from the rhizosphere extending to the soil, mycorrhizal fungi colonize and interconnect simultaneously two environments, specifically the roots of a host plant and the rhizosphere (Fig. 22.1) (Jansa et al., 2013; Drigo et al., 2013). Whereas the hyphae inside the roots are mainly surrounded by plant cells and thus exposed to a stable environment, the hyphae extending to the soil are exposed to great variations in abiotic characteristics and they constantly interact with other organisms in soil such as fungi, macrofauna, microfauna, and bacteria (Fig. 22.1) (Drigo et al., 2013; Kaiser et al., 2015). The total soil volume under the influence of mycorrhizal plants either through roots or through mycorrhizal hyphae is referred to as the mycorrhizosphere and includes the combined effects of the soil microbial communities in the rhizosphere and in the hyphosphere (Fig. 22.1) (Timonen and Marschner, 2006). Hence the mycorrhizosphere might be considered the crossroad of the root–soil habitat, where complex fine-scale gradients of substrate availability, water potential, and redox state modify the root–soil environment and consequently the composition, activity, and colonization ability of the surrounding beneficial, pathogenic, and commensal microbial communities (Fig. 22.1) (Timonen and Marschner, 2006). In past studies, using soil compartments where only hyphae could enter, Andrade et al. (1998) found that the presence of hyphae caused an increase in fluorescent pseudomonads and an Alcaligenes eutrophus strain, whereas Ravnskov et al. (1999) reported a decrease in Pseudomonas fluorescens strain DF57, and Burkholderia and Bradyrhizobium were present on

FIGURE 22.1  The mycorrhizosphere might be considered the crossroad of the root–soil habitat, where c­ omplex fine-scale gradients of substrate availability, water potential, and redox state modify the root–soil environment and consequently the composition, activity, and colonization ability of the surrounding beneficial, pathogenic, and c­ ommensal microbial community. The pathways of photosynthetically fixed carbon (C) in the below-ground ­compartment of the plant–fungi–soil system are described in this figure. Thickness of lines represents approximate volume/rate of fluxes. Respiration losses are not shown.

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the EcM hyphae associated with pine trees (Timonen and Hurek, 2006). Several other studies observed that the presence of mycorrhizal hyphae can modify the bacterial community composition and growth (Scheublin et al., 2010; Toljander et al., 2007; Mansfeld-Giese et al., 2002; Marschner and Baumann, 2003; Rillig et al., 2006; Filion et al., 1999; Cheeke et al., 2015; Bender and van der Heijden, 2015; van der Heijden et al., 2015). There is little evidence for the association of mycorrhizal hyphae with eukaryotes (i.e., yeasts) and viruses, and the reasons behind recruiting a specific microflora in the mycorrhizosphere remain mostly unclear (Bonfante and Anca, 2009; Jansa et al., 2013; Bonfante and Genre, 2015). In this chapter we provide an overview of the available scientific knowledge on the identity and putative roles of hyphae-associated microbes with respect to the mycorrhizal fungi and also to the mycorrhizal host plants. We explore the dynamics of these associations under fluctuating environmental conditions and the evolving insights to understand hyphae-associated microbes. More specifically, we analyze the potential involvement of the microbes in nutrient cycling and carbon (C) transformation in the hyphosphere. We focus mainly on the AM fungi, because they represent the most widespread mycorrhizal fungal group, forming a symbiosis with the vast majority of the land plants (Chapter 1), although examples of some EcM systems, another widely abundant mycorrhizal symbiosis (Chapter 1), are also presented.

22.2  CARBON ALLOCATION FROM MYCORRHIZAL FUNGI TO THE HYPHAE-ASSOCIATED MICROBES IN THE HYPHOSPHERE Between 4% and (apparently up to) 30% of the net plant photosynthetic production is transferred to mycorrhizal fungi and subsequently released to soil microbes (Fig. 22.1) (Paul and Kucey, 1981; Jakobsen and Rosendahl, 1990; Drigo et al., 2010; Lendenmann et al., 2011; Calderon et al., 2012; Kaiser et al., 2015). This release can be via direct exudation from the mycorrhizal hyphae or by immobilization in the extraradical mycelium of the mycorrhizal fungi (Fig. 22.1) (Drigo et al., 2012; Chapter 23). Both root exudation and transfer of C accumulated via photosynthetic activity to mycorrhizal fungi occur within a few hours in grasses and up to a few days in trees (Drigo et al., 2010, 2013; Kaiser et al., 2015). Direct evidence for the transfer of plant-derived C from mycorrhizal hyphae to their associated microbes is still lacking; however, previous studies have shown that AM fungal hyphae act as a major hub for translocating fresh plant C to soil microbes and release C gradually to certain hyphae-associated microbes such as Burkholderia spp. and Pseudomonas spp. (Figs. 22.2 and 22.3) (Johansson et al., 2004; Toljander et al., 2007; Drigo et al., 2010, 2013; Kaiser et al., 2015). This has the indirect ability to shift the hyphosphere active microbial community, which might reward AM fungi with increased nutrient availability through stimulation of soil organic matter (SOM) depolymerization (Hodge and Storer, 2015; Jansa et al., 2013). Another pathway for the photosynthates to move from the plant to the hyphae-associated microbes is through decay of mycorrhizal mycelium (Fig. 22.1). We know little about the fate of C contained in hyphal biomass once it dies (Drigo et al., 2012; Chapter 23). This is particularly important because when detached from the roots, external mycorrhizal mycelia become a large resource of both labile and recalcitrant C that can fuel the activity of hyphosphere microorganisms and contribute to the formation of SOM. For example, in a poplar plantation, EcM mycelium was the dominant pathway (62%) through which the C entered the SOM pool,

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ELEV CO2

CO2

Leaf Litter alteration in production and biochemistry

Respiration

PLANT

Plant species-specific alteration in qualitative and quantitative C allocation, plant growth and photosynthesis ROOT

C

Nutrients uptake

Organic substrates alteration in energy availability for microbial growth and maintenance

Respiration

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Mycorrhizae alteration in infection and activity

Respiration

Fine roots alteration in growth, turnover, respiration rates, nutrient uptake C

Microfauna and Soil animals alteration in composition and function (i.e. Nematodes and Protozoa)

SOIL MICROBIAL COMMUNTIES BULK

RHIZOSPHERE

Bacteria Alteration in the rhizocompetent community structure and size (i.e. Pseudomonas spp.)

Antibiotic productions alteration

Fungi Alteration in the community structure aod size

Pathogenic fungi community alteration

Alteration in composition and function of the microbial communities

Soil organic matter (SOM) alteration in chemical composition and C allocation storage

Soil Nutrient Availability

FIGURE 22.2  Conceptual model, which describes the response of a dominant plant species to elevated atmospheric carbon dioxide (ELEV CO2). The increased photosynthetically fixed carbon (C) allocation is initially directed mainly to mycorrhizas and root tissues. Mycorrhizas are translocating the C into the soil microbial communities, thereby changing the structure, size, and activity of the rhizosphere microbial communities (bacteria and fungi) to a larger extent than the communities of the bulk soil. Soil microbial communities subsequently affect food web interactions and mediate the ecosystem feedback systems that regulate the cycling of both C and mineral nutrients such as nitrogen. Adapted from Drigo, B., Kowalchuk, G.A., van Veen, J.A., 2008. Climate change goes underground: effects of elevated atmospheric CO2 on microbial community structure and activities in the rhizosphere. Biology and Fertility of Soils 44, 667–679.

exceeding the input via leaf litter and fine root turnover (Godbold et al., 2006). Use of mesh in-growth bags suggested that EcM mycelium had a mean residence time of about 10 years (Wallander and Thelin 2008), but this is likely to differ based on a number of factors including fungal species, morphology, and edaphic conditions. Both the speed at which dead mycelium decays and the factors controlling this in soils are poorly understood and the evidence is sometimes contradictory. For example, Koide and Malcolm (2009) analyses suggest a key role of the nitrogen (N) content of hyphae. Wilkinson et al. (2011) found that hyphal necromass rapidly stimulated microbial activity and that this was exaggerated by the effects of species richness of the dead fungi. This finding suggested that some nutrient resources in hyphae were being degraded preferentially by saprotrophic microbes. A similar situation has been found after additions of dissolved organic C (Cleveland et al., 2007). Lindahl et al. (2010) has also shown that saprotrophic fungi can respond rapidly to inputs of EcM mycelium. These

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FIGURE 22.3  Conceptual model of carbon (C) flow in mycorrhizal plant–soil systems summarizing the observed effects of elevated carbon dioxide (CO2) atmospheric concentrations on soil microbial communities. Brown arrows indicate increases and decreases in the respective community sizes, as determined by real-time polymerase chain reaction and lipid analyses in Drigo et al. (2007) and Drigo et al. (2010), as well as changes in community structure and C flow. Absence of an arrow indicates no significant change in the community size or structure. Red arrows indicate no effect of increased C availability because of elevated CO2 on the Actinomycetes spp. and Bacillus spp. communities. The mechanism and magnitude of the C flow along the soil food web are indicated by the green arrows. AM, Arbuscular mycorrhizal. Effects on nematodes are based on Drigo, B., Kowalchuk, G.A., Yergeau, E., Bezemer, T.M., Boschker, H.T.S., 2007. Impact of elevated C dioxide on the rhizosphere communities of Carex arenaria and Festuca rubra. Global Change Biology 13, 2396–2410, whereas the other data are based on Drigo, B., van Veen, J., Pijl, A.S., Kielak, A.M., Gamper, H., et al., 2010. Shifting C flow from roots into associated microbial communities in response to elevated atmospheric CO2. Proceedings of the National Academy of Sciences United States of America 107, 10938–10942.

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studies indicate the need to better understand the link between C inputs from mycorrhizal hyphae and the soil biodiversity and microbial activity.

22.3  INVOLVEMENT OF THE HYPHAE-ASSOCIATED MICROBES IN NUTRIENT CYCLING AND CARBON TRANSFORMATION IN THE HYPHOSPHERE Trading C implies a mutually beneficial symbiosis and whereas tracer experiments provide evidence for the flow of C from roots to bacteria via a mycorrhizal pathway (Drigo et al., 2010, 2013; Kaiser et al., 2015), the payment in the opposite direction, if any, remains largely unknown. Jansa et al. (2013) pose the question of whether hyphae-associated microbes perform functions that benefit the fungus, or whether they are “free-riders,” mopping up hyphal exudates. To establish a trading relationship with beneficial microbes, mycorrhizal fungi must first be able to recognize and interact differently with potential partners they encounter in order to avoid supplying/releasing C to free-riders or parasites. In an in vitro study of interactions between an EcM fungus (Laccaria bicolor S238 N) and three soil bacteria, the fungus responds differently to each bacterial strain 14 days after inoculation (Deveau et al., 2015). The largest shifts in L. bicolor transcriptome were in response to the antagonistic bacteria, Collimonas fungivorans, but beneficial and neutral P. fluorescens strains also induced unique transcriptional responses. All three bacteria responded to the presence of the fungus with changes in transcripts related to nutrient acquisition and metabolism, but whether this indicates trading or competition with the fungus for nutrients is unknown. Mycorrhizal hyphae shape their surrounding bacterial community, and the mycorrhizosphere microbial community is different to that found in the bulk soil and also distinct from the microbial community attached to an inert substrate (Scheublin et al., 2010). Although not direct evidence of trading, the distinct bacterial community found in the hyphosphere, the ability of fungi to discriminate between different bacteria, and the observed flow of C from fungal hyphae to bacteria in the hyphosphere set the scene for a possible trade of resources between mycorrhizal fungi and bacteria. In this section we consider what the bacteria may have to offer in exchange for the fungal C.

22.3.1 Mycorrhiza Helper Bacteria One of the most well studied interactions between mycorrhizal fungi and bacteria are those of mycorrhiza helper bacteria (MHB) (Garbaye, 1994). Garbaye describes MHB mechanisms in terms of aiding establishment of the plant–fungus symbiosis and we follow this convention, considering postinfection fungal–bacterial interactions in the latter sections. Examples of MHB in both the AM and EcM literature are plentiful and have been reviewed for example by Frey-Klett et al. (2007). Mechanisms of bacteria helping the mycorrhizal fungus to establish the symbiosis with a plant include improving germination of fungal spores and enhancement of hyphal growth preinfection, altering signaling between plant and fungus, and modifying rhizosphere soil (Garbaye, 1994), as well as modifying the plant root to improve cell permeability or increase root branching (Artursson et al., 2006). Direct evidence of transfer of C in return for these services is lacking and any transfer of plant C via mycorrhizas would be separated in time from the benefits provided by the bacteria. IV.  MYCORRHIZAL MEDIATION OF ECOSYSTEM CARBON FLUXES AND SOIL CARBON STORAGE

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22.3.2 Endocellular Bacteria A more intimate relationship exists between mycorrhizas and endocellular bacteria (Bonfante and Anca, 2009). In the case of Candidatus Glomeribacter gigasporarum, an obligate endosymbiont of the AM fungus Gigaspora margarita (Bianciotto et al., 2001; Bonfante and Anca, 2009), the loss of metabolic pathways indicates a dependence on the host fungi for energy and nutrients and thus evidence for a transfer of C from the fungi to the bacteria (Ghignone et al., 2012). In comparative transcriptomic and proteomic studies of wild-type and cured lines of G. margarita spores, the presence of the endosymbiont increased the antioxidant capacity of the fungus and affected metabolism (Salvioli et al., 2016; Vannini et al., 2016), and the wild-type line also had a higher growth rate than the cured fungal isolate (Lumini et al., 2007). Although these studies focus on the establishment stages of mycorrhizas, endosymbiont activity was greatest in the extraradical mycelia during plant–fungus symbiosis (Anca et al., 2009), with the functional significance of the endosymbiont occurrence being not entirely understood.

22.3.3 Nutrient Uptake 22.3.3.1 Phosphorus The greatest progress toward identifying a mutually beneficial trade has been made in the interactions between AM fungi and phosphorus (P)-solubilizing bacteria (PSBs). Improving P nutrition of plants is cited as a major benefit of the plant–AM fungi symbiosis. Plant P availability is limited by transformation of insoluble or moderately soluble forms of P to water-soluble orthophosphate and the rate of P diffusion through soil water (Richardson and Simpson, 2011). The extended reach of hyphae beyond the rhizosphere zone accounts for the ability of AM fungi to improve plant P status (Bolan, 1991), providing a direct route for the plant to obtain P from the bulk soil rather than relying on diffusion through the soil solution where the microbial community could interfere through competition for readily available P. However, the ability of AM fungi, or at least of Rhizophagus irregularis, to mobilize adsorbed P from solid surfaces or from organic forms is limited (Antunes et al., 2007; Tisserant et al., 2013) and the benefits of AM fungi to plant P nutrition are likely dependent on bacteria first mobilizing P from the SOM or from the solid surfaces. PSBs release P in plant available forms by producing organic acids that solubilize mineral phosphates, and acid phosphatases that release P from organically bound P compounds (Rodriguez and Fraga, 1999). PSBs are common in soil and can constitute over 40% of all the culturable rhizosphere community (Jorquera et al., 2008). The capacity to mobilize P is shared among phylogenetically diverse taxa including Gammaproteobacteria [e.g., Pseudomonas, Enterobacter and Pantoea (Jorquera et al., 2008)] and Bacilli (Rodriguez and Fraga, 1999). PSBs are active in the rhizosphere, resulting in P depletion around the root (Richardson and Simpson, 2011); mycorrhizal hyphae extend the plants’ access to P beyond this depletion zone. In addition, the hyphosphere bacterial community may have enhanced P solubilizing capacity either through an increased proportion of PSBs (Frey-Klett et al., 2007) or other bacteria improving P solubility (Taktek et al., 2015). In vitro studies have demonstrated the ability of PSBs to grow with fungal hyphae as a sole source of C. In a split plate experiment, bacteria were separated from mycorrhizal chicory roots on a minimal medium, whereas the R. irregularis extraradical mycelia were IV.  MYCORRHIZAL MEDIATION OF ECOSYSTEM CARBON FLUXES AND SOIL CARBON STORAGE

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allowed to grow between the two compartments (Taktek et al., 2015). Populations of four PSBs, including Rhizobium, Burkholderia, and Rahnella spp., grew and exhibited phosphate solubilizing activity with AM hyphae as a C source; however, the C may have been obtained through turnover of fungal hyphae. Zhang et al. (2016) collected hyphal exudates consisting of sugars (galactose, glucose, and trehalose) and carboxylates (aconitate, citrate, and, in higher P concentration media, succinate) from R. irregularis. Growth and phosphatase activity of a population of the PSBs, Rahnella aquatilis, was demonstrated in the presence of these R. irregularis exudates. These experiments show that the PSBs tightly associated with hyphae are able to grow on the AM fungi hyphal exudates as a sole nutrient source, but whether the bacteria offer anything in return to the fungi is less clear. It is also unclear whether the release of C compounds from hyphae is actively regulated by the fungi or just passively leaked. Synergistic effects of inoculation of PSBs and AM fungi on plant biomass and P content have been observed through 32P-tracer experiments (Azcón-Aguilar and Barea, 2015). For example, when a form of P not readily available for plant uptake, rock phosphate, was added to microcosms, onion plants co-inoculated with both AM fungi (R. irregularis) and a PSB strain (Bacillus subtilis) had greater shoot biomass, P, and N contents than noninoculated controls or plants inoculated with either AM fungi or PSB alone (Toro et al., 1997). Bacillus subtilis acted as an MHB, with AM colonization of the onion root increasing in its presence; in addition, co-inoculated plants had access to P that was otherwise unavailable. Conversely, in maize soil microcosms, co-inoculation of R. irregularis and a mixture of three PSBs (Pseudomonas alcaligenes, Bacillus megaterium, and R. aquatilis) did not improve plant performance (Wang et al., 2016). Plant roots and PSBs were spatially separated in soil compartments linked by fungal hyphae. Whereas inoculation of R. irregularis increased shoot biomass and P content of the plants, addition of PSBs did not further improve plant performance. Instead, in the presence of PSB, hyphal length density was reduced and, compared with PSB inoculation alone, microbial biomass P increased in the presence of the fungi. Using 13C-labeling, Wang et al. (2016) demonstrated transfer of plant C to PSBs via mycorrhizas, yet bacteria out-competed the AM fungus for P, providing no apparent benefit to the AM fungus in exchange for the increased C availability. Zhang et al. (2014a) further compared the interaction between the AM fungus R. irregularis and PSB R. aquatilis at different concentrations of organic P (phytate) in the soil. R. irregularis increased shoot P content of Medicago spp. at high P (75 mg kg−1), and acted synergistically with R. aquatilis to further increase shoot P. However, at low P, there was no further increase in shoot P in the presence of the PSB and instead the microbial biomass P increased in the presence of the AM fungus. The interactions between R. irregularis and the PSB R. aquatilis were dependent on soil conditions, with bacteria only providing services under favorable nutrient conditions, whereas under P limiting conditions, the interaction was competitive rather than mutualistic. Further studies are needed with different microbial strains/species and more complex communities to determine whether this is a widely occurring phenomenon. In contrast to AM fungi, EcM fungi are able to mobilize P by their own phosphatase ­activities and organic acid excretion. Still, PSB were enriched in the hyphosphere of L. bicolor associated with Douglas fir (Pseudostuga menziesii) (Frey-Klett et al., 2005), suggesting a ­possible trade of P for C between the EcM fungi and their associated bacterial communities.

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22.3.3.2 Other Nutrients In addition to P, mineral weathering by EcM fungi can mobilize nutrients including potassium, calcium, and magnesium (van Breemen et al., 2000; contrasting views in Chapters 2 and 3). This too may be the result of complex interactions between the fungi and associated bacteria. The population density of a weathering-competent bacterial strain of Burkholderia significantly increased in the presence of EcM fungi on Scots pine (Pinus sylvestris) seedlings (Koele et al., 2009) and this bacterial strain worked synergistically with the EcM fungus Scleroderma citrinum to increase plant uptake of magnesium. Profiling of mycorrhizosphere bacterial communities suggests other potential exchanges of nutrients for C, such as trades including sulfur (Gahan and Schmalenberger, 2015) and iron (Frey-Klett et al., 2005). Many of the bacteria found to be associated with EcM fungi at root tips have N-fixing capabilities, and though several studies provided evidence that bacterial N fixation did happen inside EcM root tips, this was only up to a very limited extent (Nguyen and Bruns, 2015). The supply of N to plants partnered by EcM fungi is obviously more dependent on the capabilities of the fungus itself to obtain N from the soil (Chalot and Brun, 1998). There is a debate regarding the potential role of AM fungi in supplying N to plants (Veresoglou et al., 2012; Hodge and Storer, 2015; Chapter 8). Fungal N requirements are high compared with those of the host plant (Johnson et al., 2015) and whereas AM fungi are efficient competitors for ammonium (NH4+) (Hodge and Storer, 2015), their ability to mobilize N from other sources is unclear. This high demand for N and limited ability to access it provides a potential niche for microbial symbionts. AM fungi alter the composition of soil decomposer communities (Nuccio et al., 2013), and release of C from hyphae coupled with uptake of NH4+ can increase decomposition by soil bacteria and mineralization of N in the hyphosphere (Hodge and Storer, 2015). The N mineralized by soil communities is then potentially transported by hyphae to the plant (Nuccio et al., 2013), but whether this can be considered trade or competition remains to be resolved. In terms of nutrient mobilization, the enzymatic activities of the mycorrhizal hyphae-associated bacteria are to a large extent shared with the rhizosphere bacteria. However, the larger surface area of mycorrhizal hyphae compared with that of roots and the larger soil volume explored by hyphal networks as compared with the root systems extend the potential contribution of these microbial traits beyond the rhizosphere depletion zone (Bolan, 1991). The common challenge remaining for all is to provide a direct evidence of uptake of nutrients by mycorrhizal hyphae released by bacteria associated with the hyphae.

22.3.4 Induced Protection of Plants Against Pathogens Several mechanisms have been proposed for AM fungi–induced protection of plants against pathogens, including competition with fungal pathogens for space and nutrients, enhanced nutrient status of the plant, and priming of plant defenses. Mycorrhizal induced resistance (MIR) refers to the priming of plant defenses after colonization, including salicylic acid (SA) and jasmonic acid (JA) pathways (Pozo and Azcón-Aguilar, 2007). Cameron et al. (2013) propose that MIR relies not only on colonization of the plant cells by the fungus but is a combination of fungi priming SA defenses and bacteria in the mycorrhizosphere priming JA defense pathways. The mycorrhizosphere provides an environment where bacteria can achieve high cell densities (Andrade et al., 1998), enabling quorum sensing to occur. Quorum sensing molecules are important in eliciting

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systemic responses in the plant and could be taken up by the plants directly either at the root surface or through mycorrhizal hyphae (Barto et al., 2012; Cameron et al., 2013). Association of AM fungi with biocontrol bacteria is an additional mechanism potentially providing protection against plant and fungal antagonists. Paenibacillus sp. B2, derived from sporocarps of Glomus mosseae, inhibited growth of several pathogenic fungi including Phytopthora nicotianae and reduced root damage to tomato plants (Budi et al., 1999). This ­isolate also acted as an MHB, increasing colonization of roots by G. mosseae, and the AM fungi and bacteria worked synergistically, reducing root necrosis caused by P. nicotianae more when added together than either of the organism separately (Budi et al., 1999). Other organisms with biocontrol potential such as P. fluorescens (Toljander et al., 2006; Viollet et al., 2011) and Streptomyces sp. (Scheublin et al., 2010) are reportedly enriched in the mycorrhizosphere. Although biocontrol bacteria are often effective against fungal pathogens and can also inhibit mycorrhizal colonization levels of the roots, AM fungi are not always adversely affected (Paulitz and Linderman, 1991). The nature of these interactions may be genotype or strain specific. AM fungi can be subject to mycoparasitism, such as that of Glomus intraradices by Trichoderma harzianum T-203 (Rousseau et al., 1996). Whether biocontrol bacteria can protect the mycorrhizal hyphae themselves remains an area yet to be explored experimentally.

22.3.5 Other Hyphae-Associated Microbial Benefits Horizontal transfer of bacterial genes involved in carbohydrate metabolism has been detected in the EcM species of the genus Amanita (Chaib De Mares et al., 2015). This genetic resource provides opportunities for the fungus to utilize new alternative substrates and thus colonize new niches. Although there is evidence of numerous transfers of bacterial genes that enhance the SOM degradation capacity to soil fungi, Amanita is thus far the only mycorrhizal example described of horizontal gene transfer from bacteria (Zhang et al., 2014b). The distinct bacterial communities associated with mycorrhizas are not necessarily involved in C trading. Hyphae can aid dispersal of bacteria, enabling them to cross air-filled soil pores (Kohlmeier et al., 2005; Simon et al., 2015). Other bacteria that do receive C might do so to the detriment of the fungus; for example, oxalic acid released by EcM fungi may attract the mycophagous bacteria Collimonas (Rudnick et al., 2015). Still, the C does flow from mycorrhizas to the bacteria and there are a number of services bacteria potentially provide in return. Whether the interactions between mycorrhizas and bacteria are mutually beneficial trades, competition or parasitism likely depends on the species or strains present. Interactions between the same species may have different outcomes, and whether this is dependent on the particular strains in question or the experimental design requires further investigation because the nature of the interactions between mycorrhizal fungi and associated bacteria is sensitive to the context, such as nutrient background in the soil (Ding et al., 2014; Zhang et al., 2016).

22.4  DYNAMICS OF THE MYCORRHIZOSPHERE ASSOCIATIONS UNDER FLUCTUATING ENVIRONMENTAL CONDITIONS Evidence that the climate is changing is overwhelming. Regardless of mitigation progress, atmospheric carbon dioxide (CO2) levels will probably surpass the 500 ppm threshold IV.  MYCORRHIZAL MEDIATION OF ECOSYSTEM CARBON FLUXES AND SOIL CARBON STORAGE

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by 2050, held by many as indicative of “dangerous” interference (Monastersky, 2013). Thus it is unavoidable that terrestrial ecosystems will be confronted with higher atmospheric CO2 in the future (Berrang-Ford et al., 2011). Under the condition of elevated CO2, AM fungi usually respond by increasing colonization of plant roots (Figs. 22.2 and 22.3) (Johnson and Gehring, 2007; Drigo et al., 2008, 2010), rapidly acquiring recent photosynthates (Johnson et al., 2003; Carillo et al., 2016) and significantly contributing to both fast and slow pools of the soil organic C through the retention of C in the mycelium (Olsson and Johnson, 2005; Johnson et al., 2013; Meier et al., 2015; Chapters 23 and 24). This significantly influences the hyphae-associated microbial community dynamics in the mycorrhizosphere (Fig. 22.3) (Drigo et al., 2010, 2013; Cotton et al., 2015). A shift from bacterial- to more fungal (including AM fungi)-dominated soil food webs has been observed a number of times and may prove to be a general response to elevated CO2 (Figs. 22.2 and 22.3) (Rillig and Allen, 1999; Olsson and Johnson, 2005; Drigo et al., 2007, 2010; Pritchard, 2011; Drigo et al., 2013; Treseder, 2016). This stimulation of AM fungal activity could be beneficial for the ecosystem functioning because AM fungi may play a role in maintaining soil structure (Rillig et al., 2002), soil C stabilization (Treseder and Allen, 2000; Drigo et al., 2010), and nutrient immobilization through hyphal translocation (Frey et al., 2000). However, the extent to which mycorrhizas benefit from atmospheric CO2 enrichment will probably depend heavily on the nutrient status of the plant and the fungus, and the AM fungi species present in the ecosystem (Treseder, 2016). Increases in atmospheric CO2 are thought to be largely responsible for increases in global mean surface temperatures, which are projected to increment by another 1.4–5°C by 2100 (IPCC, 2014). Such increases in temperature would further contribute to more than a 20% reduction in mean annual precipitation globally and altered patterns of rainfall, including more extreme events. Thus in the future, plants will likely experience increases in acute heat and drought stress, which can impact soil microbial function, dynamics, and biodiversity (Thomas et al., 2004; Evans and Wallenstein, 2014). Indeed, rapid rhizosphere microbial responses to sudden moisture availability, such as those resulting from rewetting events after extended periods of drought, often result in immediate mineralization of C- and N-containing compounds that accumulate during drought periods (Barnard et al., 2013). There is evidence that rhizosphere microbial communities may become more tolerant to drought when previously exposed to variable rainfall patterns through changes in microbial life history traits (Evans and Wallenstein, 2014). However, this may also influence the functional traits of the local microbial communities with potential implications for ecosystem functioning (Evans and Wallenstein, 2014). Tolerating moisture stress implies resource and nutrient investment in exopolysaccharide and spore production, and the accumulation of compatible solutes (reviewed in Schimel et al., 2007), which may become restricted as the water-film thickness is diminished by desiccation or by an abrupt flush upon rewetting dry soils (Barnard et al., 2013; Vries et al., 2012). AM fungi can help plants and soil microbes deal with drought and extreme rainfall patterns by acting, directly or indirectly, on plant functionality at the above- and below-ground levels (Chapter 17). In the plant leaves and roots, the osmotic stress caused by drought is balanced by mycorrhiza through biochemical changes that mostly imply increased biosynthesis of metabolites (i.e., proline and sugars) that act as osmolytes (Brunner et al., 2015). These metabolites help decrease the plant osmotic potential and allow the plant to sustain the cell physiological activity (Brunner et al., 2015). The AM plants withstand drought-induced oxidative stress by the increased IV.  MYCORRHIZAL MEDIATION OF ECOSYSTEM CARBON FLUXES AND SOIL CARBON STORAGE

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production of antioxidant enzymes (Rapparini and Penuelas, 2014) and the creation of a highly functional root system for nutrient/water uptake (Hodge and Storer, 2015). At the same time, AM fungal hyphae in the rhizosphere constitute an efficient pathway for nutrient/water uptake and transport, allowing a more efficient exploitation of the water and nutrient reservoirs in the soil pores where only fungal hyphae can grow, thereby bypassing the zones of water and nutrient depletion around the roots and the mycorrhizosphere microbial communities (Rapparini and Penuelas, 2014). Molecular mechanisms initiated by AM fungi to respond to drought events include gene activation for production of functional proteins such as the membrane transporter aquaporin, ion, and sugar transporters (Li et al., 2012a). In addition, AM fungi might reinforce the resistance of associated microbes to drought by improving soil structural stability that in turn increases the retention of soil water (Medina and Azcon, 2010). The response of mycorrhiza and associated soil microbial communities to climate change under field conditions, however, remains difficult to measure and is not predictable from single-factor experiments (Nielsen and Ball, 2015). Therefore multifactorial experiments are urgently needed to define ecologically relevant taxonomic and functional units for microorganisms and, consequently, improve the relevant models’ reliability (Albert et al., 2011; Chapter 26).

22.5  UNRESOLVED QUESTIONS ON TRADING CARBON AND NUTRIENT BETWEEN MYCORRHIZAS AND HYPHAE-ASSOCIATED MICROBES Mycorrhizas are in the front line of plant-soil–microbial interactions. Mycorrhizas and their associated microbes’ growth, nutrient uptake, and release of solutes vary both in space and time, and interact with heterogeneous soil microenvironments that provide habitats for associated microbes at various scales. Despite tremendous progress in method development in the past decades, finding a suitable experimental set-up to research processes occurring at the dynamic conjunction of the mycorrhizosphere still represent a major challenge. Recent methodological developments in rhizosphere research, involving visualization of two- or three-dimensional rhizosphere processes via chemical imaging, microbial imaging, and noninvasive imaging has a significant potential in visualizing the mycorrhizosphere processes across a range of scale from centimeter to submicrometers. Another recent methodological development is the use of isotopically labeled compounds with stable isotopes, such as 2H [(the stable hydrogen isotope deuterium), 13C, 15N, and ­oxygen (18O), in combination with complementary techniques such as mass spectrometry, ­high-throughput sequencing, nanoscale secondary ion mass spectrometry (NanoSIMS), and neutron ­radiography to trace the movement of water and C- and/or N-containing compounds through the ­mycorrhizosphere. These approaches have great potential to unravel mechanisms of uptake, s­ torage, and translocation of C, N, and other nutrients at the ­mycorrhizosphere level. In ­particular, NanoSIMS combines unprecedented spatial ­resolution, sensitivity, and mass ­specificity to quantitatively visualize fine-scale differences in ­metabolism and cell–cell ­interactions (­Pett-Ridge & Weber, 2011). The combination of DNA and RNA ­stable-isotope ­probing ­techniques with NanoSIMS and fluorescent in situ hybridization (FISH) will

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c­ ontribute to map the m ­ ycorrhizosphere microbial associations and reveal their function in trading nutrients. Indeed, these will allow tracking metabolic activities of single mycorrhizal ­hyphae-associated microbial cells by imaging natural isotopic/elemental composition or ­isotope distribution after ­stable-isotope probing. NanoSIMS with FISH is a particularly valuable combination for e­ xpanding our understanding of ­microbial relationships and nutrient flows in ­environmental microbial assemblages and characterizes microbial-mediated biogeochemical processes. NanoSIMS can further be combined with immunolabeling or quantitative molecular imaging to identify ­microbial communities and chemical exchanges within microbial energy webs. The use of single-cell Raman spectra might aid to detect intrinsic chemical composition of living organisms in the mycorrhizosphere as signatures or “fingerprints” (Li et al., 2012b). The combination of stable-isotope probing techniques with Raman spectroscopy has been proven to be a valuable novel approach in microbiology, microbial ecology, and clinical research. The use of stable-isotope probing–Raman spectroscopy will allow tracking C, N, and/or oxygen flows in the complex microfauna and macrofauna of the mycorrhizosphere in a quantitative and nondestructive manner, providing crucial information on the biochemical pathways that lead to the synthesis of the basic biomolecules. Although each of the presented techniques on their own provide important new insights, we believe that particularly the combined use of different methodological approaches will be promising in shedding more light onto the great number of yet unrevealed processes at the root-mycorrhiza–soil interface.

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IV.  MYCORRHIZAL MEDIATION OF ECOSYSTEM CARBON FLUXES AND SOIL CARBON STORAGE