Redistribution of soil water by a saprotrophic fungusenhances carbon mineralizationAlexander Guhr1, Werner Borken, Marie Spohn, and Egbert Matzner

Department of Soil Ecology, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, 95448 Bayreuth, Germany

Edited by Susan E. Trumbore, Max Planck Institute for Biogeochemistry, Jena, Germany, and approved October 8, 2015 (received for review July 22, 2015)

The desiccation of upper soil horizons is a common phenomenon,leading to a decrease in soil microbial activity and mineralization.Recent studies have shown that fungal communities and fungal-based food webs are less sensitive and better adapted to soildesiccation than bacterial-based food webs. One reason for a betterfungal adaptation to soil desiccation may be hydraulic redistributionof water by mycelia networks. Here we show that a saprotrophicfungus (Agaricus bisporus) redistributes water frommoist (–0.03MPa)into dry (–9.5 MPa) soil at about 0.3 cm·min−1 in single hyphae,resulting in an increase in soil water potential after 72 h. The increasein soil moisture by hydraulic redistribution significantly enhanced car-bon mineralization by 2,800% and enzymatic activity by 250–350% inthe previously dry soil compartment within 168 h. Our results dem-onstrate that hydraulic redistribution can partly compensate waterdeficiency if water is available in other zones of the mycelia network.Hydraulic redistribution is likely one of themechanisms behind higherdrought resistance of soil fungi compared with bacteria. Moreover,hydraulic redistribution by saprotrophic fungi is an underrated path-way of water transport in soils and may lead to a transfer of water tozones of high fungal activity.

saprotrophic fungi | hydraulic redistribution | drought |carbon mineralization

Drought is one of the most important and frequent abioticstresses in terrestrial ecosystems (1). With respect to soil

processes, soil desiccation limits microbial activity and decreasessoil enzyme activity (2), carbon mineralization (3, 4) and nitrogenmineralization (5). In addition, drought can also alter soil micro-bial community composition (2, 6).During desiccation and dry periods, soil fungal communities

and fungal-based food webs are better adapted to drought thanbacterial communities and bacteria-based food webs (7, 8).Bacteria are more strongly restricted than fungi (9), as bacterialactivity needs a constant supply of water (10). One reason forthe better adaptation of fungi compared with bacteria to lowsoil water potentials is seen in their strong cell walls, preventingwater losses (1). The strength of fungal cell walls can even beenhanced by cross-linking of polymers and thickening understress. Another reason for the better adaptation of fungi to soildesiccation might be hydraulic redistribution of water by my-celia networks. Hydraulic redistribution is defined as the pas-sive transport of water in soils through organisms along agradient in soil water potential and was first observed for plantroots (11). Hydraulic redistribution through plant roots im-proves plant survival and nutrient uptake by extending the lifespan and activity of roots (12) and by favoring decomposition ofsoil organic matter (13). Mycorrhiza fungal hyphae can alsorelocate water along gradients in soil water potential (12, 14–16). In addition, some studies reported the transport of nutri-ents and water over larger distances (>1 m) by saprotrophicfungal hyphae in nonsoil systems. Further, water leakage fromhyphae into dry growth medium was observed (17). The watertransport in hyphae was attributed to gradients in osmoticpotentials (18–22).Saprotrophic fungi are main regulators of soil nutrient cycling,

litter decomposition, and soil respiration due to their specific

enzymatic activities (23, 24) and due to the high density of hy-phae in soil (up to 800 m·g−1 soil) (25), and especially in litterlayers. The ability of saprotrophic fungi to distribute water wouldprovide a direct and fast connection between water and nutrientsources in soils that would be hardly accessible to bacteria. Thiscould have an enormous impact on decomposition processesunder drought conditions.Here, we show the potential of the saprotrophic fungus Agaricus

bisporus for hydraulic redistribution and impact of water redistri-bution on carbon mineralization in a desiccated soil.

Results and DiscussionWe analyzed hydraulic redistribution using 4–5 replicates of two-chamber units filled with homogenized mineral soil. The singlechambers of each unit were separated by a 2-mm air gap toprevent bulk flow of water (Fig. S1). After inoculation and theestablishment of hyphal bridges through the air gap between thetwo chambers, the soil in both chambers was desiccated to a soilwater potential of about –9.5 MPa. Thereafter, only chamber Iwas rewetted to field capacity (–0.03 MPa), whereas chamber IIof each unit remained dry. Hydraulic redistribution was pre-vented in the controls by cutting the hyphal bridges between thetwo chambers. Volumetric water contents, soil water potential,and deuterium-labeling were used for quantification of hydraulicredistribution.At 72 h after rewetting chamber I with deuterium-labeled

water, the volumetric soil water content of chamber II increasedon average by about 0.02 cm3 H2O cm−3 soil. The small increaseof 0.006 cm3 H2O cm−3 soil in chamber II of the controls can beattributed to diffusion of gaseous water through the air gap.Deuterium signatures of redistributed soil water supported thedetermined rates of hydraulic redistribution by A. bisporus. After72 h, the amount of water redistributed from chamber I to chamberII was three times higher with hydraulic redistribution comparedwith the controls (Fig. 1). This was accompanied by an increase inwater potential of the bulk soil in chamber II from –9.5 to –6.9 MPa

Significance

This work shows a mechanism behind the observed higherdrought resistance of soil fungi compared with bacteria. It alsodemonstrates the relevance of hydraulic redistribution bysaprotrophic fungi for ecosystem ecology by influencing thecarbon and water cycle in soils and terrestrial ecosystems un-der drought. Furthermore, we documented a so far underratedpathway of water in desiccated soils.

Author contributions: A.G., W.B., M.S., and E.M. designed research; A.G. performed re-search; A.G. analyzed data; and A.G., W.B., M.S., and E.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this article have been deposited in Dryad DigitalRepository, datadryad.org (doi:10.5061/dryad.bm56k).1To whom correspondence should be addressed. Email: alexander.guhr@uni-bayreuth.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1514435112/-/DCSupplemental.

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with hydraulic redistribution, whereas in the controls soil waterpotential only increased from –9.2 to –8.2 MPa.After 72 h, hydraulic redistribution through hyphae had resulted

in an average water flux of 67 μL·cm−2·d−1 between both chambers.The number of hyphae bridging the two chambers in the air gap wasabout 2,300 cm−2. The total water flux between the chambers cor-responds to a specific water flux of about 0.03 μL·d−1 through asingle hyphae. A flow velocity of about 0.3 cm·min−1 in single hy-phae was calculated using the Bernoulli’s equation for the centralcell lumen of the hyphae (outer diameter, 4.4 μm; central cell lu-men, ∼2/3 of the total hyphal diameter) (26). This corresponds toflow velocities observed for the central cell lumen in arbuscularmycorrhizal hyphae (0.3 cm·min−1) (27).In general, passive mass flow is considered as the main mecha-

nism of water transport in fungal hyphae (28), but active transportmechanisms for water might also be involved, such as cytoplasmicstreaming (with velocities of 0.03–0.05 cm·min−1 in hyphae) (29),vesicles moved by motor proteins (up to 0.02 cm·min−1) (30),and vacuolar pathways (about 0.005 cm·min−1) (31). Given theseflow velocities, active transport cannot be the reason for the highvelocities observed here, and thus, water redistribution is driven bypassive mass flow along the soil water potential gradient. The massflow of water can be apoplastic as well as symplastic. Symplasticwater transport at such flow velocities requires the presenceof aquaporins. Those are likely part of the cell membrane inA. bisporus as the encoding genes were identified (32).Amount and velocity of hydraulic redistribution by sapro-

trophic fungi might even be higher under natural soil conditionsthan in our experiment. First, hyphal density in the chamber ex-periment was small compared with natural soils. From the numberof hyphae in the air gap and an estimated tortuosity factor of 2 forhyphal length in soil pores, a total hyphal length of about 30 m·g−1

soil dry weight close to the air gap was calculated. In natural soils,hyphal lengths of up to 800 m·g−1 soil are reported (25). Second,the potential for hydraulic redistribution is likely larger forfungal species that form more complex mycelial structures,such as rhizomorphs or cords—that is, aggregations of longitu-dinal aligned hyphae. Although A. bisporus is capable of formingsuch cords, only single hyphae was found in our experiment. Cord-forming fungi were found to be very effective in translocatingnutrients (21, 22, 33, 34). The transport of nutrients in cords ismuch faster than in nondifferentiated mycelium (28), and thedistances for nutrient translocation in fungal cords can be >1 m

(22). After localization and identification of substrates in the soil,cord connections are strengthened to exploit the substrate whileother parts of the mycelium regress (35, 36), preferentially directingwater to the substrate. Hence, as with nutrient translocation,hydraulic redistribution through fungal cords and especiallyrhizomorphs is probably more effective than through single hyphae.In an additional experiment (Fig. S2), we determined the effect

of hydraulic redistribution on carbon mineralization in desiccatedsoils by measuring soil enzyme activities and mineralization of13C-labeled plant material. Soil zymography allowed us to measureenzyme activity in situ under different soil water contents, thusshowing the effect of soil moisture on enzyme activity. Hydraulicredistribution by hyphae increased enzyme activity on average by350% for N-acetylglucosaminidase and by 250% for cellobiohy-drolase compared with the controls (Fig. 2). Enzyme activitiesdecreased with increasing distance to the air gap in chamber IIwhen hydraulic redistribution was active (Fig. 3), whereas no suchpattern was observed in the controls. Similar relations of soilenzyme activities were observed between soils with and withoutirrigation (2, 37), emphasizing the significance of hydraulic redis-tribution by fungi.Hydraulic redistribution led to an enrichment in 13C of the

respired CO2 that was apparent already after 24 h and increasedthroughout the rest of the experiment. After 168 h, the cumu-lative C mineralization amounted to 59.7 g CO2·kg

−1 C withactive hydraulic redistribution and 2.1 g CO2·kg

−1 C in controls(Mann–Whitney U test, U1,9 = 0, P < 0.01; Fig. 4). This relationis similar to variations in C mineralization rates between dry andrewetted litter in forest soils (0.11 and 137 mg CO2 kg

−1 C h−1,respectively) (38), which again illustrates the significance of hy-draulic redistribution for mineralization.Therefore, saprotrophic fungi not only have the capability to

redistribute water but can also partly compensate unfavorablesoil moisture conditions in desiccated soil as long as water isavailable in other zones of the mycelial network, like in deepersoil horizons. Under dry conditions, desiccation descends fromthe substrate-rich upper soil layers to the subsoil. The leakage ofwater from hyphae into the surrounding soil is concentrated tohyphal tips (39), and hyphal tips are concentrated in the growingpart of the mycelium close to the substrate (35). Water re-distribution through the mycelium, bypassing capillary transportthrough soil pores, has probably been underrated as a pathway ofwater movement in desiccated soils. Hydraulic redistribution is

Fig. 1. Hydraulic redistribution (HR) by hyphae. Amount of water redis-tributed from chamber I to chamber II 72 h after the irrigation of chamber I.Calculation based on hydrogen stable isotope ratios. Black, active hydraulicredistribution; gray, control with no fungal connection (mean + SEM, n = 4;Mann–Whitney U test, U1,7 = 0, **P < 0.01).

Fig. 2. Enzyme activity on the soil surface. Measured in chamber II, 7 d afterirrigation of chamber I. Black, active hydraulic redistribution (HR); gray,control with no fungal connection (mean + SEM, n = 5; NAG, lme, F1;39 =20.30, **P < 0.01; cellobiohydrolase, lme, F1,39 = 15.91, **P < 0.01; NAG,N-acetylglucosaminidase).

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of particular relevance for bridging capillary barriers. Overall,hydraulic redistribution likely leads to a transfer of water tohotspots of fungal activity in dry soils with preferential wetting ofthe surrounding substrate. It enables high fungal enzymatic ac-tivity in the growth zone even under low soil water potentials.Hence, hydraulic redistribution likely is one of the mechanismsbehind the higher resistance of soil fungi compared with bacteriato soil desiccation.

Materials and MethodsGeneral Setup. Experiments were carried out in mesocosms (adapted from 39)(Fig. S1) represented by two chambers (a 6 × 20 × 15 cm), filled with a steamsterilized mixture (2:1:1 vol/vol/vol) of loamy soil (17% clay; 76% silt; 7%sand) and medium coarse quartz sand (Dorsilit 8; particle size range, 0.3–0.8 mm) as well as coarse quartz sand (Dorsilit 7, 0.6–1.2 mm; Dorfner GmbH& Co.). The two chambers made of Makrolon (Bayer AG) had openings onthe sides facing each other, which were covered by two 160-μm pore sizestainless steel mesh screens. A 2-mm-thick air gap between both chambers

prevented capillary flow of water and was stabilized by two additionalperforated stainless steel mesh screens with 2 mm pore sizes. Chamber topswere removable and were air-tight if closed. The soil surface was com-pressed slightly to obtain a uniformly flat surface. Fungal cultures [DSM No.3056, A. bisporus (Lange) Imbach] were received from the Leibniz InstituteDSMZ and grown on malt extract peptone agar at 14 °C. The soil of chamberI was inoculated by placing a 1 cm2 agar plate with fungal hyphae into thesubstrate close to the air gap at a depth of approx. 2 cm. A. bisporus waschosen because it is one of the best studied filamentous fungal species, witha complete available genome, that shows a fast growth rate. Both chamberswere maintained at 23 °C and irrigated regularly to field capacity with aliquid fungal growth medium (2% glucose, 0.2% peptone, 0.2% yeast ex-tract, 0.1% K2HPO4, 0.46% KH2PO4, and 0.05% MgSO4) (40) for 6 wk. Thechamber tops were kept open but were covered with glass microfiber filterpaper (Grade 934-AH, Whatman Ltd.) during the growth phase to facilitateair exchange and avoid contaminations. Volumetric water content wascontrolled continuously using soil moisture sensors monitoring the dielectricconstant of the media (ECH2O-10 moisture sensor; Decagon Devices Inc.).

Quantification of Hydraulic Redistribution. After desiccation for 6 wk at 23 °Cto a soil water potential of approximately –9.5 MPa in both chambers,chamber I was rewetted to field capacity (–0.03 MPa) with deuterium-labeled water (3% at deuterium enrichment; ROTH GmbH + Co. KG). Mes-cosms were then closed air-tight and only opened for sampling of soil cores.Soil cores of chamber II were destructively sampled 72 h after irrigation ofchamber I. Water for isotope analyses was extracted from soil samples bycryogenic vacuum extraction (41). Hydrogen-stable isotope analyses wereconducted at the Laboratory for Isotopic-Biogeochemistry (University ofBayreuth) using thermal conversion/isotope-ratio mass-spectrometry (isotopemass spectrometer, delta V advantage; Thermo Fisher Scientific).

In addition, soil water potential was measured on collected soil samples(4 cm diameter, 0.5 cm thickness) using the chilled mirror dewpoint method(WP4-T; Decagon Devices Inc.) (42).

Controls were established by mesocosms treated in the same way as de-scribed above, but the hyphal connections between the chambers in the airgap were severed by cutting with a thin stainless-steel wire before irrigatingchamber I. In total, four mescosms with intact fungal connections as well asfour control mescosoms were treated with deuterium-labeled water.

Mineralization of Organic Matter. CO2 efflux from the soil is an indicator ofthe general activity of soil microorganisms and was therefore used to esti-mate the impact of hydraulic redistribution on mineralization of organicmatter under drought conditions. The use of 13C-labeled plant material(Triticum aestivum L. green shoots; >97 atom% 13C; C/N ratio, 15; IsoLife)enabled us to trace the origin of collected CO2. Labeled plant material (fiveground samples of 20 mg each) were placed at regular intervals of 4 cm fromthe mesh screen on the soil surface of the nonirrigated chamber II, shortlybefore rewetting chamber I. Mescosms were then closed air-tight and werenot opened for 7 d. CO2 effluxes were regularly measured for 7 d at 20 °C

Fig. 3. Enzyme activity on the soil surface. (A) Cellobiohydrolase and (B)N-acetylglucosaminidase in chamber II of one mesocosm with active hy-draulic redistribution, 7 d after irrigation of chamber I. The calibration linefor the enzyme activity is presented at the bottom.

Fig. 4. Cumulative carbon mineralization. Calculation based on 13C-CO2

efflux from labeled plant material in chamber II, following irrigation ofchamber I. Black, active hydraulic redistribution (HR); gray, control with nofungal connection (solid lines, mean values; dashed lines, SEM; n = 5).

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using the dynamic closed-chamber technique (43) (Fig. S2). CO2 concentrationsin the mesocosms headspace (1.2 L) were measured every 6 min for 30-minperiods for the first 24 h with an infrared gas analyzer (LiCOR 820; Licor).Beginning with the second day of experiments, CO2 concentrations weremeasured for periods of 48 min. Soil CO2 effluxes were calculated from theslope of the linear regression between CO2 concentration and incubation time.An alternative air path flow was opened at the end of each measurementcycle for 30 min on the first day of measurements and subsequently for 12 minto flush the system with CO2-free synthetic air and reduce the CO2 concen-tration. In addition, the extracted air was collected every 12 h for furtheranalysis of 13C isotope contents to determine the percentage of decomposedplant material in chamber II. Extracted air was stored in 10 mL butyl rubberseptum-capped vials by flushing for 90 s. Septa were heated at 105 °C for 12 hbefore vial closing to prolong stability of CO2 isotope composition (44). Vialswere flushed with N2 for 90 s prior to use to remove environmental CO2.13C-CO2 efflux for the whole measurement cycle was interpolated from mea-sured 13C-CO2 efflux values using a Gaussian function extended with a linearterm to adjust for divergences from a normality distribution. Function fitnesswas optimized using Solver (Microsoft Cooperation).

δ13C analyses were conducted at the Laboratory for Isotopic-Biogeochemistry(University of Bayreuth) using an Elemental analyzer (NA 1108; CE Instruments)–isotope ratio mass spectrometer (delta S; Finnigan MAT) linkage.

In total, fivemescosmswith intact fungal connections aswell as five controlmescosoms were treated with labeled plant material.

Analysis of Soil Enzyme Activity. The impact of hydraulic redistribution on soilenzyme activity was analyzed using soil zymography (ref. 45 modified by ref.46). This in situ method allows for analysis of the 2D distribution of enzymeactivities in soil with high spatial resolution and under different watercontents in contrast to more traditional methods that are based on thedetermination of enzyme activity in solution. Hence, this provided a com-prehensive picture of the allocation of redistributed water in chamber II andthe resulting influence on enzyme activities. In addition, the study of enzy-matic activities provides functional information on specific aspects of organicmatter decomposition and can therefore support the results of CO2 effluxmeasurements. N-acetylglucosaminidase and cellobiohydrolase activity wereanalyzed using the artificial substrates 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (4-MNG) and 4-methylumbelliferyl β-D-cellobioside (4-MC;both Sigma-Aldrich Chemie GmbH), respectively. The fluorogenic 4-methylumbelliferone (MUF) is released from 4-MNG and 4-MC due to hydrolyticcleavage in the presence of compatible enzymes. In soils, the activity of chiti-nase is considered as a good indicator of fungal biomass and activity (47, 48).

A 1% agarose gel (size of 0.1 × 12.0 × 11.0 cm) was cast in systems usuallyused for vertical gel electrophoresis (Biometra GmbH). The gel was sliced infour parts at 2 × 11 cm, and all four parts were attached to the soil surface ofthe nonirrigated chamber II in the space between the labeled plant materialsamples. Polyamide membrane filters (0.45 μm pore size; Sartolon, SartoriusAG) were sliced in 10 parts at 2 × 11 cm. Half of the slices were saturatedwith a 4-MNG solution or 4-MC solution (25% wt/vol; Sigma-Aldrich Chemie

GmbH), respectively. Four slices of each group were placed in turn on top ofthe gel slices, starting with the 4-MNG group. The membrane filter wasextracted after an incubation time of 25 min at 20 °C for 4-MNG and 20 minfor 4-MC and illuminated on a fluorescent transilluminator in the dark(wavelength, 355 nm; Desaga GmbH). Pictures were taken with a digitalcamera (Nikon D3100) and analyzed in comparison with controls withouthydraulic redistribution. To adjust for differences in exposure time, whichare necessary to avoid overexposure at high activities and loss of details atlow activities, one filter slice was not incubated on the soil but photo-graphed together with the others and served as a standard of zero activity.A calibration line was prepared from membranes soaked in different solu-tions of MUF concentrations (0, 35, 70, 130, and 200 μM). These calibrationmembranes were cut into strips of 2 cm and photographed under the UVlight in the same way as the zymogram membranes. The amount of MUF onan area basis was calculated from the volume of solution taken up by themembrane and by the size of the membrane.

Image processing and analysis were done using the open source softwareimageJ 1.46r (Wayne Rasband, National Institutes of Health, Bethesda, MD).The digital images were transformed to 8-bit—that is, grayscale images. Toillustrate the results, the values of the grayscale image were depicted in falsecolor. The linear correlation between the MUF concentration and the meanof grayscale in an area of 4 cm2 of each calibration gel were calculated usingthe software R. A segment at 1.5 × 7.5 cm with no visible disturbance wasselected from the soil zymograms, and mean values of the grayscale weremeasured. Values were standardized based on the difference between thestandards of zero activity and the calibration membrane with 0 μM MUFconcentration. Values were expressed as pmol MUF/h and mm2.

Data Analysis. All statistical analyses and graphics were done using R 3.1.0(R Developmental Core Team). Normality and homogeneity of the data weretested using Shapiro–Wilk test and Levene’s test, respectively. Enzyme ac-tivities were analyzed using linear mixed-effect models as implemented inthe R package nlme (49). The sample origin from the different chambers wasadded as a random factor into the model to adjust for random variancesamong chambers. For pair-wise post hoc comparisons, general linear hy-potheses based on Tukey all-pair comparisons were conducted, using theR package multcomp (50).

Kruskal–Wallis test with pair-wise Wilcox tests for post hoc comparisonswere used if data were not normal and/or variances were not homoge-neously distributed.

ACKNOWLEDGMENTS. We thank C. Werner and M. Dubbert for their supportfor the cryogenic vacuum extraction of soil samples, G. Rambold and theMycology Department of the University of Bayreuth for help with and usageof facilities for treating and storing fungal cultures, and B. Gilfedder for criticalfeedback and discussions. Furthermore, we thank the Central IsotopicLaboratory of the Bayreuth Center of Ecology and Environmental Research(BayCEER) for the stable isotope analyses. This study was supported byDeutsche Forschungsgemeinschaft Grant DFG-MA1089/23-1.

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