Edith C. Hammer

Tit for tat? A mycorrhizal fungus accumulates phosphorus under low plant carbon availability

The exchange of carbohydrates and mineral nutrients in the arbuscular mycorrhizal (AM) symbiosis must be controlled by both partners in order to sustain an evolutionarily stable mutualism. Plants downregulate their carbon (C) flow to the fungus when nutrient levels are sufficient, while the mechanism controlling fungal nutrient transfer is unknown. Here, we show that the fungus accumulates nutrients when connected to a host that is of less benefit to the fungus, indicating a potential of the fungus to control the transfer of nutrients. We used a monoxenic in vitro model of root organ cultures associated with Glomus intraradices, in which we manipulated the C availability to the plant. We found that G. intraradices accumulated up to seven times more nutrients in its spores, and up to nine times more in its hyphae, when the C pool available to the associated roots was halved. The strongest effect was found for phosphorus (P), considered to be the most important nutrient in the AM symbiosis. Other elements such as potassium and chorine were also accumulated, but to a lesser extent, while no accumulation of iron or manganese was found. Our results suggest a functional linkage between C and P exchange.

Elemental composition of arbuscular mycorrhizal fungi at high salinity.

We investigated the elemental composition of spores and hyphae of arbuscular mycorrhizal fungi (AMF) collected from two saline sites at the desert border in Tunisia, and of Glomus intraradices grown in vitro with or without addition of NaCl to the medium, by proton-induced X-ray emission. We compared the elemental composition of the field AMF to those of the soil and the associated plants. The spores and hyphae from the saline soils showed strongly elevated levels of Ca, Cl, Mg, Fe, Si, and K compared to their growth environment. In contrast, the spores of both the field-derived AMF and the in vitro grown G. intraradices contained lower or not elevated Na levels compared to their growth environment. This resulted in higher K:Na and Ca:Na ratios in spores than in soil, but lower than in the associated plants for the field AMF. The K:Na and Ca:Na ratios of G. intraradices grown in monoxenic cultures were also in the same range as those of the field AMF and did not change even when those ratios in the growth medium were lowered several orders of magnitude by adding NaCl. These results indicate that AMF can selectively take up elements such as K and Ca, which act as osmotic equivalents while they avoid uptake of toxic Na. This could make them important in the alleviation of salinity stress in their plant hosts.

Plants as resource islands and storage units - adopting the mycocentric view of arbuscular mycorrhizal networks

The majority of herbaceous plants are connected by arbuscular mycorrhizal (AM) fungi in complex networks, but how this affects carbon (C) and phosphorus (P) allocation among symbionts is poorly understood. We utilized a monoxenic AM system where hyphae from donor roots colonized two younger receiver roots of varying C status. AM fungal C allocation from donor to receiver compartments was followed by measuring the (13)C contents in fungal- and plant-specific lipids, and P movement from a hyphal compartment was traced using (33)P. Four times more (13)C was translocated from donor to C-limited receiver roots, but C remained in fungal tissue. Root C status did not influence the overall AM colonization, but arbuscule density was twice as high in non-C-limited roots, and they received 10 times more (33)P. The number of hyphal connections between compartments did not influence C and P allocation. Interestingly, there were more fungal storage lipids, but fewer structural lipids inside C-limited roots. Our results indicate that AM colonization may poorly reflect host quality as C can be supplied from neighboring roots. A mycocentric view of the symbiosis is proposed where C-delivering hosts are resource islands for the exchange of P for C, and C-limited hosts are storage units.

Do arbuscular mycorrhizal fungi stabilize litter‐derived carbon in soil?

1. Fine roots and mycorrhiza often represent the largest input of carbon (C) into soils and are therefore of primary relevance to the soil C balance. Arbuscular mycorrhizal (AM) fungi have previously been found to increase litter decomposition which may lead to reduced soil C stocks, but these studies have focused on immediate decomposition of relatively high amounts of high-quality litter and may therefore not hold in many ecological settings over longer terms. 2. Here, we assessed the effect of mycorrhizal fungi on the fate of C and nitrogen (N) contained within a realistic amount of highly C-13-/N-15-labelled root litter in soil. This litter was either added fresh or after a 3-month incubation period under field conditions to a hyphal in-growth core where mycorrhizal abundance was either reduced or not through rotation. After 3 months of incubation with a plant under glasshouse conditions, the effect of turning cores on residual C-13 and N-15 inside the cores was measured, as well as C-13 incorporation in microbial signature fatty acids and N-15 incorporation of plants. 3. Turning of cores increased the abundance of fungal decomposers and C-13 loss from cores, while N-15 content of cores and plants was unaffected. Despite the difference in disturbance that turning the cores could have caused, the results suggest that mycorrhizal fungi and field incubation of litter acted to additively increase the proportion of C-13 left in cores. 4. Synthesis. Apart from stimulating litter decomposition as previously shown, mycorrhizas can also stabilize C during litter decomposition and this effect is persistent through time. (Less)

Phosphorus Availability Influences Elemental Uptake in the Mycorrhizal Fungus Glomus intraradices, as Revealed by Particle-Induced X-Ray Emission Analysis

ABSTRACT We investigated element accumulation in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal spores and mycelia growing in monoxenic cultures were analyzed. The elemental composition was quantified using particle-induced X-ray emission (PIXE) in combination with scanning transmission ion microscopy. In the spores, Ca and Fe were associated mainly with the spore wall, while P and K showed patchy distributions and their concentrations were correlated. Excess of P in the hyphal growth medium increased the P and Si concentrations in spores and increased the K/Ca ratio in spores. Increased P availability decreased the concentration of Zn and Mn in spores. We concluded that the availability of P influences the uptake and accumulation of several elements in spores. It is demonstrated that PIXE analysis is a powerful tool for quantitative analysis of elemental accumulation in fungal mycelia.

Arbuscular mycorrhizal fungi – short‐term liability but long‐term benefits for soil carbon storage?

The interaction between plants and mycorrhizal fungi represents a major link between atmospheric and soil-contained carbon (C). In order to estimate the fate of atmospheric CO2 under the projected increases in the upcoming century, ranging from an increase of 20% to > 200% compared with current concentrations (Pachauri & Reisinger, 2007), it is crucial to understand how plants and mycorrhizal fungi either buffer or exacerbate atmospheric CO2 rises through their effects on soil C sequestration. Indirect evidence suggests that arbuscular mycorrhizal fungi (AMF) generally stimulate soil carbon pools (Wilson et al., 2009), and experience enhanced growth under elevated CO2 (eCO2) (Antoninka et al., 2011), leading to the assumption that they will buffer atmospheric CO2 increases. However, long-term experiments under eCO2 show both increased carbon storage (Iversen et al., 2012) and accelerated decomposition (negating the effect of the increase of soil carbon inputs; Phillips et al., 2012), leaving the question as to whether soils will buffer against CO2 increases wide open.While there is a dearth of direct empirical evidence regarding the involvement of AMF in soil C storage processes under conditions of global change, there is uncertainty about how component processes leading to soil C storage will be affected. Recently, Cheng et al. (2012) presented a compelling body of evidence to suggest that AMF may diminish rather than enhance soil C pools in the topsoil. Their findings are based on the observation that, in the presence of AMF, fresh above-ground plant litter decomposes faster, in particular at eCO2 and increased nitrogen (N) concentrations (Cheng et al., 2012). This observation suggests that AMF can accelerate decomposition and can even lead to a loss of soil C pools, at least in the short term. However, we feel that other parts of the soil C equation will need to be resolved in order to fully understand how AMF affect long-term soil C-sequestration potential. This is because short-term experiments do not account for potential increases in organic matter (OM) of plant or microbial origin triggered by increased decomposition; long-term (decadal scale) effects of soil biota such as AMF can be qualitatively different from short-term effects; and pulse increases of CO2 and N affect soils in a way that may not represent a system where CO2 and N are at consistently higher concentrations. Soil C sequestration is the net build-up of C in the entire soil profile through accumulation ofOM from plant, fungal (and other microbial) and animal origins. Decomposition of OM is an ongoing process, and snapshot rate assessments must therefore be interpreted with caution. If a particular nutrient (e.g. C or N) is elevated, this may lead to accelerated decomposition, but conclusions about soil C gain or loss can only be drawn if the effect of biomass increases of all biota is also incorporated into the equation (Fig. 1a). This becomes apparent in a simplemodel where AMF are allowed to produce recalcitrant compounds (such as various polysaccharides (K€ogel-Knabner, 2002) and glomalin, in line with experimental observations; Rillig, 2004) that contribute to the future OM fraction (see Fig. 1b). In the short term, an AMFmediated increase in decomposition of labile plant littermay lead to a reduction of soil C. However, the C balance is offset by a longterm gain in recalcitrant compounds (Fig. 1a). Contributions of AMF are likely to be further amplified through physically protecting OM from decomposition by means of soil aggregation (Rillig, 2004) and via a general increase in plant productivity and hence significantly higher litter input (Hoeksema et al., 2010). The principal mechanism by which AMF are proposed to stimulate soil C efflux is through priming of decomposers, which is a commonly observed soil-biotic response to increased (labile) OM deposition (de Graaff et al., 2010). However, whether this stimulation of soil saprobes is a permanent effect will require further study: C pulses and the resulting soil fungal community responses are a well-appreciated side-effect of sudden-onset CO2 exposure designs, which disappearwhenCO2 is gradually increased (Klironomos et al., 2005). Such sudden increases in atmospheric CO2 concentration are unlikely to happen in the near future. By contrast, other parameters will likely change under permanently altered amounts of resources, for instance litter quality. Decomposability of plant litter is known to decrease following plant exposure to eCO2 (Norby et al., 2001), and has the potential to buffer soil C concentrations against effects predicted from shortterm experiments. Thus the magnitude of priming effects through AMF under permanent eCO2 (as opposed to pulse elevation) must be scaled against indirect effects on litter quality to fully appreciate the contribution of AMF to plant-derived soil C concentrations. A way in which short-term experimental studies could control for some of these effects is to include additional treatments where soil and OM (thus controlling for factors such as soil aggregation and quantity and quality of litter) have been preconditioned, to the extent feasible, according to experimental treatments of interest (e.g. ambient vs eCO2; low vs high N; + vs AMF) in a factorial manner. Another highly useful addition might be a treatment where plant roots but not AMF can access plant litter generated under ambient vs eCO2 concentrations (a true nonAMF treatment). Even though these approaches do not resolve all fundamental issues arising from predicting long-term processes with short-term experiments, decomposition in the eCO2 and AMF treatments can be compared between ‘uniform’ and ‘preconditioned’ (according to treatment) plant and soil material. This way

The Influence of Different Stresses on Glomalin Levels in an Arbuscular Mycorrhizal Fungus—Salinity Increases Glomalin Content

Glomalin is a glycoprotein produced by arbuscular mycorrhizal (AM) fungi, and the soil fraction containing glomalin is correlated with soil aggregation. Thus, factors potentially influencing glomalin production could be of relevance for this ecosystem process and for understanding AM fungal physiology. Previous work indicated that glomalin production in AM fungi may be a stress response, or related to suboptimal mycelium growth. We show here that environmental stress can enhance glomalin production in the mycelium of the AM fungus Glomus intraradices. We applied NaCl and glycerol in different intensities to the medium in which the fungus was grown in vitro, causing salinity stress and osmotic stress, respectively. As a third stress type, we simulated grazing on the extraradical hyphae of the fungus by mechanically injuring the mycelium by clipping. NaCl caused a strong increase, while the clipping treatment led to a marginally significant increase in glomalin production. Even though salinity stress includes osmotic stress, we found substantially different responses in glomalin production due to the NaCl and the glycerol treatment, as glycerol addition did not cause any response. Thus, our results indicate that glomalin is involved in inducible stress responses in AM fungi for salinity, and possibly grazing stress.

Elemental composition in vesicles of an arbuscular mycorrhizal fungus, as revealed by PIXE analysis

We investigated element accumulation in vesicles of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, extracted from the roots of inoculated leek plants. The elemental composition (elements heavier than Mg) was quantified using particle-induced X-ray emission (PIXE), in combination with scanning transmission ion microscopy (STIM). In vesicles, P was the most abundant of the elements analysed, followed by Ca, S, Si and K. We analysed 12 vesicles from two root systems and found that the variation between vesicles was particularly high for P and Si. The P content related positively to Si, Zn and K, while its relation to Cl fitted to a negative power function. Vesicle transects showed that P and K were present in central parts, while Ca was present mainly near the vesicle surfaces. The results showed that P is an important part (0.5% of the dry weight) of the vesicle content and that the distribution of some elements, within mycelia, may be strongly correlated.

The interplay between P uptake pathways in mycorrhizal peas: a combined physiological and gene‐silencing approach

Arbuscular mycorrhizal fungi (AMF) have a key role in plant phosphate (Pi) uptake by their efficient capture of soil phosphorus (P) that is transferred to the plant via Pi transporters in the root cortical cells. The activity of this mycorrhizal Pi uptake pathway is often associated with downregulation of Pi transporter genes in the direct Pi uptake pathway. As the total Pi taken up by the plant is determined by the combined activity of mycorrhizal and direct pathways, it is important to understand the interplay between these, in particular the actual activity of the pathways. To study this interplay we modulated the delivery of Pi via the mycorrhizal pathway in Pisum sativum by two means: (1) Partial downregulation by virus-induced gene silencing of PsPT4, a putative Pi transporter gene in the mycorrhizal pathway. This resulted in decreased fungal development in roots and soil and led to reduced plant Pi uptake. (2) Changing the percentage of AMF-colonized root length by using non-, half-mycorrhizal or full-mycorrhizal split-root systems. The combination of split roots, use of ³²P and ³³P isotopes and partial silencing of PsPT4 enabled us to show that the expression of PsPT1, a putative Pi transporter gene in the direct pathway, was negatively correlated with increasing mycorrhizal uptake capacity of the plant, both locally and systemically. However, transcript changes in PsPT1 were not translated into corresponding, systemic changes in actual direct Pi uptake. Our results suggest that AMF have a limited long-distance impact on the direct pathway.

Phosphorus and carbon availability regulate structural composition and complexity of AM fungal mycelium.

The regulation of the structural composition and complexity of the mycelium of arbuscular mycorrhizal (AM) fungi is not well understood due to their obligate biotrophic nature. The aim of this study was to investigate the structure of extraradical mycelium at high and low availability of carbon (C) to the roots and phosphorus (P) to the fungus. We used monoxenic cultures of the AM fungus Rhizophagus irregularis (formerly Glomus intraradices) with transformed carrot roots as the host in a cultivation system including a root-free compartment into which the extraradical mycelium could grow. We found that high C availability increased hyphal length and spore production and anastomosis formation within individual mycelia. High P availability increased the formation of branched absorbing structures and reduced spore production and the overall length of runner hyphae. The complexity of the mycelium, as indicated by its fractal dimensions, increased with both high C and P availability. The results indicate that low P availability induces a growth pattern that reflects foraging for both P and C. Low C availability to AM roots could still support the explorative development of the mycelium when P availability was low. These findings help us to better understand the development of AM fungi in ecosystems with high P input and/or when plants are subjected to shading, grazing or any management practice that reduces the photosynthetic ability of the plant.