Plant Soil (2012) 355:63–73 DOI 10.1007/s11104-011-1080-8

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Changes in soil hyphal abundance and viability can alter the patterns of hydraulic redistribution by plant roots

José Ignacio Querejeta & Louise M. Egerton-Warburton & Iván Prieto & Rodrigo Vargas & Michael F. Allen

Received: 13 June 2011 /Accepted: 22 November 2011 /Published online: 9 December 2011 # Springer Science+Business Media B.V. 2011

Abstract Results Contrary to expectations, both upper soil and Background and aims We conducted a mesocosm receiver seedlings contained significantly greater pro- study to investigate the extent to which the process portions of HLW in mesocosms where the abundance of of hydraulic redistribution of soil water by plant roots mycorrhizal hyphal links between donor and receiver is affected by mycorrhizosphere disturbance. roots had been sharply reduced by fungicide application. Methods We used deuterium-labeled water to track the Reduced soil hyphal density and viability likely ham- transfer of hydraulically lifted water (HLW) from pered soil moisture retention properties in fungicide- well-hydrated donor oaks (Quercus agrifolia Nee.) to treated mesocosms, thus leading to faster soil water drought-stressed receiver seedlings growing together depletion in upper compartments. The resulting steeper in mycorrhizal or fungicide-treated mesocosms. We soil water potential gradient between taproot and upper hypothesized that the transfer of HLW from donor to compartments enhanced hydraulic redistribution in receiver plants would be enhanced in undisturbed fungicide-treated mesocosms. (non-fungicide-treated) mesocosms where an intact Conclusions Belowground disturbances that reduce soil mycorrhizal hyphal network was present. hyphal density and viability in the mycorrhizosphere can

Responsible Editor: Angela Hodge.

José Ignacio Querejeta, Louise M. Egerton-Warburton and Iván Prieto contributed equally to this work.

J. I. Querejeta (*) I. Prieto Departamento de Conservación de Suelos y Aguas, Estación Experimental de Zonas Áridas-Consejo Superior Centro de Edafología y Biología Aplicada del de Investigaciones Científicas (EEZA-CSIC), Segura-Consejo Superior de Investigaciones La Cañada de San Urbano, Científicas (CEBAS-CSIC), Almería, Spain Campus Universitario de Espinardo, 30100 Murcia, Spain R. Vargas e-mail: [email protected] Departamento de Biología de la Conservación, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Mexico L. M. Egerton-Warburton Chicago Botanic Garden, M. F. Allen 1000 Lake Cook Road, Center for Conservation Biology, University of California, Glencoe, IL, USA Riverside, CA, USA 64 Plant Soil (2012) 355:63–73 alter the patterns of hydraulic redistribution by roots HL favors the maintenance of fine root function in dry through effects on soil hydraulic properties. soil (Caldwell et al. 1998;Espeletaetal.2004;Bauerle et al. 2008) and plays a crucial role in the direct transfer Keywords Hydraulic lift . Water redistribution . of water from roots to associated mycorrhizal fungal Mycorrhizal fungi . Quercus agrifolia . Soil water symbionts (Querejeta et al. 2003). This last process retention properties . Mycorrhizosphere disturbance can prolong the lifespan of mycorrhizal fungal hyphae in dry soil (Querejeta et al. 2007a, 2009), which may in Abbreviations turn improve the water and nutrient status of the host HLW Hydraulically lifted water plant (Egerton-Warburton et al. 2008). Furthermore, HL Hydraulic lift shallow-rooted plants growing within the rhizosphere CMN Common mycorrhizal network of a deep rooted plant conducting HL can benefit from δD Deuterium this process, thus leading to water parasitism among EMF Ectomycorrhizal fungi neighboring plants (Caldwell 1990; Ludwig et al. AMF Arbuscular mycorrhizal fungi 2003;Prietoetal.2011). For instance, Dawson (1993) found that shallow rooted plants growing near a large maple tree conducting HL were able to use up to 60% of hydraulically lifted water (HLW), and thereby showed Introduction higher stomatal conductance and photosynthetic rate than those with no access to HLW. Hydraulic redistribution is the passive movement of There are two main pathways by which HLW from water from wet to dry soil layers through plant root deep soil layers can be taken up by shallow-rooted systems (Burgess et al. 1998). Water potential gradients plants: either directly from soil after water efflux from in the soil profile provide the driving force for this the roots of a neighboring deep-rooted plant, or via process, and determine its direction and magnitude common mycorrhizal networks (CMNs) connecting the (Scholz et al. 2008). This phenomenon is termed roots of donor and receiver plants. Egerton-Warburton et “hydraulic lift” (HL) when water moves upward from al. (2007) used deuterium-labeled water and fluorescent deep, wet soil layers to upper, drier layers (Richards and tracers to demonstrate that ectomycorrhizal and arbuscu- Caldwell 1987). Hydraulic redistribution can also occur lar mycorrhizal extraradical hyphae provide a potential in a downward direction, from wet shallow to drier, pathwayforthetransferofHLWbetweenplantssharing deeper soil layers, (e.g., after a rain event; Burgess et common mycorrhizal networks. In their study, the trans- al. 2001; Hultine et al. 2003; Schulze et al. 1998), or fer of water between well-hydrated donor plants and laterally within the plant root system (Smart et al. 2005). drought-stressed receiver plants appeared to be driven Efflux of water from roots to soil usually takes place by source-sink relationships mediated by water potential during the night when plant transpiration is minimal gradients similar to those that drive the process of HL. (Caldwell and Richards 1989; Richards and Caldwell Thus, when the shallow rooted receiver becomes a sink 1987).HLhasbeenreportedinawidearrayofplant during periods when the upper soil is dry, HLW may taxa ranging from small grasses to large trees (Caldwell move from deep, wet soil layers via donor roots and et al. 1998), and in a wide variety of ecosystems, from associated mycorrhizal mycelium into the receiver plant. arid and semi-arid environments (Richards and Caldwell Redistribution of water via CMNs, and/or HLW efflux 1987; Yoder and Nowak 1999; Armas et al. 2010; Prieto from donor roots to soil followed by subsequent uptake et al. 2010) to mesic temperate environments (Emerman by receiver roots, represent two alternative pathways for and Dawson 1996; Kurz-Besson et al. 2006), tropical inter-plant water transfer that are not mutually exclusive forests (Meinzer et al. 2004; Moreira et al. 2003;Scholz (Plamboeck et al. 2007;Warrenetal.2008). However, et al. 2008), and even mangroves (Hao et al. 2009). the relative contributions of these two pathways in the HL exerts multiple beneficial effects on plant water transfer of HLW between neighboring plants has not balance, such as enhancing transpiration during dry been investigated so far. periods and delaying the onset of plant drought In the present study, our primary goal was to assess stress (Caldwell and Richards 1989;Ryeletal. the impact of mycorrhizosphere disturbance (i.e., fungi- 2002;Caldwelletal.1998; Meinzer et al. 2004). cide addition) on hydraulic redistribution of soil water Plant Soil (2012) 355:63–73 65 by plant roots. More specifically, we wanted to Germinated acorns were inoculated by repeated addition determine whether changes in the abundance and of suspensions of spores of the ectomycorrhizal fungi viability of soil hyphae might influence the pat- Pisolithus tinctorius (Pers.) Coker & Couch and Sclero- terns of hydraulic redistribution during moderate derma sp. (Mycorrhizal Applications Inc., Grants Pass, drought. We also investigated the relative contri- Oregon) suspended in deionized water (total 15×108 butions of each pathway (root–soil–root pathway, spores per mesocosm). All mesocosms were irrigated or redistribution through CMNs) to the transfer of to field capacity twice a week for 22 months. The HLW between donor and receiver plants during control plants were watered with a Hoagland nutrient mild drought. We used deuterium-labeled water to solution so that they would achieve a size similar to that track the transfer of HLW from well-hydrated, of the inoculated plants at the end of the experiment. deep-rooted donor oaks to drought-stressed receiver Greenhouse temperatures were maintained at 18–24°C oak seedlings growing together in mycorrhizal or (night/day; October to May), and 22–35°C (June– fungicide-treated mesocosms. We hypothesized that October). The 20 mm air gap between upper and receiver seedlings would contain a greater propor- taproot compartments was initially filled with soil tion of HLW in mesocosms where an intact CMN to allow for root growth and extension into the taproot was present than in mesocosms where mycorrhizal compartment. Seventeen months after mesocosm estab- hyphal links between donor and receiver oaks had lishment, thick woody roots were observed bridging the been severely damaged by fungicide application (i.e., gap between upper and taproot compartments. The soil mycorrhizosphere disturbance). filling the gap was then washed and removed. Eighteen months after mesocosm establishment, coast live oak seedlings (2 cm tall and <10 cm of Materials and methods taproot length) were excavated and transplanted into the upper root compartments of the mesocosms to Mesocosm establishment serve as receiver plants (three receiver seedlings per mesocosm). Receiver oak seedlings were obtained Quercus agrifolia Nee. (California coast live oak) from a nearby nursery. Previous studies have shown donor plants were grown in two-compartment meso- that naturally regenerating and nursery-grown oak seed- cosms comprising a lower taproot compartment and an lings are extensively colonized by both ectomycorrhizal upper compartment separated from each other by an air and arbuscular mycorrhizal fungi (Egerton-Warburton barrier to prevent capillarity and mass flow of water and Allen 2001). Therefore transplanted oak seedlings between them (for further details, see Querejeta et al. most likely introduced arbuscular, ectomycorrhizal and 2003). Mesocosms were made of transparent acrylic saprophytic fungi to all the mesocosms (including non- plate 6 mm thick. The dimensions (width × width × inoculated mesocosms) height) of upper root compartments were 10×15× 30 cm, whereas the cylindrical taproot compartments Fungicide application were 8×30 cm (diameter × height). A 20 mm air gap separated upper compartment from taproot compart- Twenty months after mesocosm establishment, the fun- ment in all mesocosms. Both compartments were filled gicide Fludioxinil was applied to four mesocosms (the with a steam-sterilized mixture of a loamy soil, coarse ones which had been left uninoculated at the onset of the sand, and fine sand (1:1:1 by volume) with pH06.8, experiment). The goal was to reduce mycorrhizal colo- −1 KCl-extractable N (NO3+NH4)04 μgg , and HCO3- nization of roots and the abundance of mycorrhizal extractable P09 μgg−1. Stratified acorns of Q. agrifolia mycelium in the soil. The remaining (preinoculated) were surface sterilized (10% v/v HClO4,10min), seven mesocoms were left undisturbed. Fludioxinil is a germinated in moist vermiculite, and planted into phenylpyrrole fungicide that provides broad spectrum the upper compartments of the mesocosms (one donor activity against Ascomycetes, Deuteromycetes, and oak per mesocosm). A total of 11 replicate mesocosms Basidiomycetes by inhibiting mycelial growth. A sus- were established in a positive pressure greenhouse, of pension of Fludioxinil (50% wettable powder) was pre- which 7 were inoculated with ectomycorrhizal fungi, pared (80 mg per liter of deionized water), and each of and 4 were left un-inoculated and served as controls. the four uninoculated mesocosm received 200 ml of this 66 Plant Soil (2012) 355:63–73 suspension. Fungicide application to uninoculated Sampling and laboratory procedures mesocosms was repeated five successive times over a period of 50 days. At dawn of the fifth day after irrigation withdrawal from In order to determine whether fungicide application upper root compartments, we collected stem sections might alter the water holding capacity of soil, we from donor oaks, whole aboveground biomass from placed 70 g of the same soil used for the experiment receiver seedlings and bulk soil samples from upper root in 11 ceramic funnels. Six of the funnels were watered compartments in all the mesocosms. Water for hydrogen with deionized water (control) and the remaining five stable isotope analyses was extracted from plant and soil were watered with fungicide solution (Fludioxinil samples using a cryogenic vacuum distillation line 50% wettable powder; 80 mg per liter of deionized (Ehleringer and Osmond 1989). Hydrogen stable iso- water) until water was observed to drip down the tope analyses were conducted at the Stable Isotope funnel tip. Water was then allowed to drain until Facility of the Department of Earth and Planetary constant weight was achieved. When this occurred Sciences, University of New Mexico. Hydrogen (24 h) soils were weighed. Then, soils were placed in isotope ratios were measured using a continuous an oven at 90°C until constant weight (24 h) was flow high temperature reduction technique (Sharp et al. reached. Soil water retention capacity (WRC,%) for 2001). Briefly, 1 μL aliquots of water are injected into a each treatment (control or fungicide-treated) was calcu- helium stream through a heated septum. The vaporized lated by weight difference as: sample is reduced to H2 and CO while passing through a graphite column heated to 1450°C. Reactant gases are ðg g Þ WRCð%Þ¼ saturated dry purified by passage through a gas chromatography gdry column, through a Finnegan MAT CONFLO II inter- There were not significant (t-test00.78; P00.46) face/open split for helium dilution, and into a Finnegan differences in moisture retention capacity between soil MAT Delta XL Plus mass spectrometer. Data are ‰ treatments, with mean soil water retention capacities of reported in conventional delta notation, defined as 52.15±1.38% and 53.45±0.73% for fungicide-treated deviation from an internationally accepted reference and control treatments, respectively. Therefore, we con- standard (VSMOW: Vienna standard mean ocean water). δ ‰ cluded that fungicide addition by itself does not change D measurements had a precision of ±2 .Weuseda the moisture retention capacity of soil. simple two-end-member linear mixing model (Dawson et al. 2002) to calculate the proportion of deuterium-labeled water used by donor oaks. The two end members were Application of deuterium-labeled water tap water (δD0−62.3‰) and deuterium labeled water (δD01,000‰). Twenty-two months after mesocosm establishment, the Soil water potential measurements were conducted upper compartments of all mesocosms were irrigated to on freshly collected soil samples from upper root com- field capacity (600 ml of water added), after which partments, using the chilled mirror dewpoint method irrigation to upper compartments was withheld for 5 days (CX-2, Decagon Devices, Pullman WA, Gee et al. to create the necessary soil water potential gradient for 1992). hydraulic redistribution to occur. During this 5-day peri- Root samples were sieved from freshly collected od, soil in taproot compartments was maintained at or bulk soil samples from upper compartments, washed near saturation by frequent (three times per day) irriga- free of adhering soil, and fine roots (≤1 mm) were tion with deuterium-labeled water. Deuterium-labeled hand-picked from each sample. A sub-sample of roots tracer water was prepared by adding 1.65 ml of pure was stained using Trypan blue (Koske and Gemma

D2O (99.8% deuterium enrichment, Sigma Chemical 1989), and evaluated for percent colonization by AMF Co.) to 10 l of tap water (δD0−62.3‰). The resulting and EMF using the modified line intersect method deuterium-labeled water used to irrigate the taproot com- (McGonigle et al. 1990). Extramatrical hyphae were partments had a δD≈1,000‰. Deuterium-labeled water extracted from 10 g duplicate sub-samples of soil from has been used as an effective tracer of HLW movement each soil core using a modification of the procedure of in many studies (e.g., Peñuelas and Filella 2003; Frey and Ellis (1997) followed by vital staining to Egerton-Warburton et al. 2007; Plamboeck et al. 2007). determine the lengths of live fungal hyphae present Plant Soil (2012) 355:63–73 67 in each location. Direct immunofluorescence with Statistical analyses antibodies raised against spores of four of the major AMF genera was used to evaluate AMF viability, All statistical analyses were conducted using the SPSS since the immune reaction will only proceed with live 13.0 program. Data were log-transformed when neces- hyphae (Allen et al. 1999). For each sub-sample, sary to ensure homoscedasticity. Plant, fungal and soil 500 μL aliquots of hyphal suspension were placed variables were analyzed by Student’s T test and/or into each of five microfuge tubes followed by 100 μl Mann–Whitney U test to detect significant differences of an individual antiserum of the four major AMF genera (P<0.05) between treatments. Regression analyses (Scutellospora, Gigaspora, Acaulospora, Glomus) con- among measured plant, fungal and soil variables were jugated to FITC (fluorescein isothyocianate); 100 μlof conducted across treatments to assess the effects of deionized water was added to the fifth tube as a control mycorrhizosphere disturbance (i.e., fungicide addition) for fungal autofluorescence. Samples were incubated on hydraulic redistribution. overnight at room temperature, and then filtered and rinsed with deionized water over a membrane, and mounted in glycerol. All samples were viewed under Results fluorescence microscopy (Zeiss Axioskop 2) using a FITC filter combination (excitation 475–490 nm, mirror Mean donor oak size was not significantly different 505 nm, emission 503–535nm)andscoredforthe between undisturbed mesocosms (height, 93±12 cm) presence or absence of FITC-labeled hyphae. Hyphal and fungicide-treated mesocosms (87±13 cm) at time counts were taken in at least 100 fields of view per slide of sampling. The size of receiver seedlings was also (× 400), and the length of hyphae was calculated and not significantly different between undisturbed (10.1± converted to meters of hyphae per gram of soil (Tennant 0.7 cm) and fungicide-treated (9.8±0.7) mesocosms at 1975). Data were averaged over all four AMF genera for that time. each sample. Percent ectomycorrhizal colonization of roots in The length of viable EMF hyphae was determined on upper compartments was significantly lower (P< sub-samples incubated with fluoroscein diacetate 0.001) in fungicide-treated mesocosms (43.94±4.1%) (FDA). FDA staining is typically used to detect than in undisturbed mesocosms (74.27±3.1%). Nine metabolically-active fungal hyphae since a quantitative different ectomycorrhizal morphotypes were found in relationship exists between the percentage of FDA- undisturbed mesocosms, whereas only seven EMF stained hyphae and mycelial growth rates (Söderström morphotypes were encountered in fungicide-treated 1977). Metabolically-active hyphae assimilate and mesocosms. Based on morphological characters, over hydrolyze FDA to fluoroscein, which fluoresces green, 50% of EMF roots appeared to be formed by Sclero- whereas non-viable hyphae do not fluoresce. An FDA derma sp. in both treatments (thus indicating mycor- stock solution was prepared by dissolving 5 mg FDA in rhizal contamination of non-inoculated mesocosms in 1 mL acetone and adding 0.1 M phosphate buffer (pH the greenhouse). By contrast, less than 10% of EMF 7.4) to give a final concentration of 50 μgl−1. A 500 μl roots appeared to be formed by Pisolithus sp. All other aliquot of the hyphal suspension was incubated with an EMF morphotypes must have been introduced in the equal amount of FDA stock solution for 5 min at room mesocosms by seedling transplantation from the temperature and then immediately observed and scored nursery. Presence of AMF in the mesocoms was also in a Zeiss Axioskop 2 using fluorescence and a filter likely the result of seedling transplantation from the combination suitable for FITC. Fifty random fields of nursery. Percent arbuscular mycorrhizal colonization of view per slide were scored for live hyphae using the roots in upper compartments was not significantly dif- gridline intersect method and converted to hyphal length ferent (P>0.05)between fungicide-treated (4.02±0.60%) per dry mass soil (Tennant 1975). It should be noted that and undisturbed mesocosms (3.32±1.05%). although specific groups of fungi can differ in the extent Soil hyphal length in the upper compartments of of FDA staining (Söderström 1977), the FDA test does undisturbed mesocosms averaged 70.63±7.72 m g−1 not clearly distinguish among the different groups of (total), of which 46.74±5.84 m g−1 was viable hyphae. fungi present in an individual sample, such as mycor- About 25% of total hyphal length and 14% of viable rhizal versus saprotrophic fungi. hyphal length could be attributable to arbuscular 68 Plant Soil (2012) 355:63–73 mycorrhizal fungi in the undisturbed mesocosms. The remaining hyphae appeared to be mostly EMF, although it should be noted that visual differentiation between EMF and saprophytic hyphae based on morphological traits is extremely problematic. Applica- tion of fungicide reduced the abundance of total and viable hyphae in upper compartments by 54% and 93%, respectively (Fig. 1). Mean soil hyphal length in fungicide-treated mesocosms was 32.68±3.01 m g−1, of which only 3.06±1.00 m g−1 was viable. Mean soil water potential in upper compartments remained above −1.2 MPa in all mesocosms at time of sampling (after a 5-day-long drying cycle), but was sig- nificantly lower in fungicide-treated than in undisturbed mesocosms (Fig. 1) despite same size of oaks in both treatments. Across treatments, soil water potential in upper compartments was strongly positively correlated with viable hyphal length in soil (R200.78; P00.003). At time of sampling, donor oaks were using deuterium-labeled water from taproot compartments in both treatments, as indicated by strong deuterium enrichment of their stem water in all mesocosms (Fig. 2a). However, deuterium enrichment of stem water was significantly greater (P00.017) in fungicide-treated than in undisturbed donor oaks, which indicates greater

Fig. 2 Hydrogen isotope composition of water extracted from a) donor oak stems, b) soil in upper compartments, and c) aboveground biomass of receiver seedlings in fungicide-treated (Fungicide treated, N04) or undisturbed mesocosms (Undisturbed, N07). Asterisks indicate significant differences between treat- ments at P<0.05

utilization of water from taproot compartments in the former. Across treatments, deuterium enrichment of stem water in donor oaks was negatively related to viable hyphal abundance (R200.484; P00.017) in upper root compartments, and marginally negatively related to total hyphal abundance (R200.331; P00.064). Analysis of the hydrogen isotope composition of soil water in upper compartments showed deuterium enrichment above background levels (irrigation water δD0−62.3‰) in all mesocosms. However, soil water in upper compartments was significantly more Fig. 1 Total and viable hyphal length densities (upper) and soil enriched in deuterium (P<0.05) in fungicide-treated water potential in the upper compartments of fungicide-treated than in undisturbed mesocosms (Fig. 2b), thus indicat- 0 (Fungicide treated, N 4) or undisturbed mesocosms (Undisturbed, ing that a greater proportion of upper soil water content N07). ** indicates significant differences between treatments at P< 0.01. * indicates significant differences at P<0.05 (Mann–Whitney was HLW in the former. Across treatments, deuterium U test) enrichment of soil water in upper compartments was Plant Soil (2012) 355:63–73 69 negatively related to both viable hyphal length (R20 water in fungicide treated mesocosms than in undis- 0.596; P<0.01) and soil water potential (R200.681; turbed mesocosms. It is important to note that a P00.002). non-limiting supply of deuterium-labeled water Deuterium enrichment of shoot water in receiver was available to donor oak roots in the taproot seedlings was significantly higher in fungicide-treated compartments of both treatments. At constant soil than in undisturbed mesocosms (Fig. 2c), which indi- water potentials near saturation in taproot compart- cates that the receiver seedlings contained a greater pro- ments, the gradient between compartments would portion of HLW in mesocosms where soil hyphae were be greater at lower soil water potentials in upper less abundant and viable. Across treatments, deuterium compartments, hence generating a stronger driving enrichment of shoot water in receiver seedlings was force for HL. This explains why soil water potentials positively related to deuterium enrichment of soil water and deuterium enrichment of soil water in upper com- in upper compartments (R200.44; P00.027). Shoot wa- partments were negatively correlated with each other ter in receiver seedlings was about 10‰ more enriched across treatments. in deuterium than upper soil water in both treatments The greater proportion of deuterium-labeled HLW in (Fig. 2b, c), due to evaporative isotopic enrichment of the upper compartments of fungicide treated mesocosms foliage water in seedlings. than in undisturbed mesocosms must have been the result of: a) more highly enriched deuterium-labeled HLW leaking out of donor oak roots into upper com- Discussion partment soil; b) greater quantities of deuterium-labeled HLW leaking out of donor oak roots; c) deuterium- Repeated fungicide application over a period of 50 days labeled HLW mixing with a smaller volume of residual effectively reduced the density and viability of soil soil water in upper compartments; or, most likely, d) a hyphae, as well as the proportion of roots colonized by combination of all these effects. In the absence of gravi- ectomycorrhizal fungi in upper compartments. There- metric soil water content data, it is unfortunately not fore fungicide application to the mesocosms (i.e., myco- possible to conduct mass balance calculations to pre- rhizosphere disturbance) allowed us to evaluate the cisely determine the exact amounts of deuterium-labeled influence of soil hyphal density and viability on the HLW that were lifted from taproot compartments to patterns of hydraulic redistribution during moderate upper root compartments in each treatment. drought. The potential role of mycorrhizal hyphae in facili- We hypothesized that greater mycorrhizal hyphal tating the transfer of HLW from donor oaks to receiver density and viability in rhizosphere soil would facilitate seedlings was overshadowed by the stronger influence HLW transfer from donor oaks to seedlings. Contrary to of differences in the magnitude of soil water potential expectations, receiver seedlings used a greater propor- gradients between treatments. Importantly, differences tion of HLW in disturbed mesocosms where the abun- in deuterium enrichment between upper soil water and dance of mycorrhizal hyphal links between donor and shoot water in receiver seedlings were very similar in receiver roots had been sharply reduced by fungicide both treatments (Fig. 2b, c). This result does not sup- application. Furthermore, a greater proportion of port a direct hyphal pathway for the transfer of HLW deuterium-labeled water was taken up from taproot from donor to receiver plants under mild drought con- compartments and redistributed to upper soil by donor ditions. Instead, our data support a stronger role of the oaks in fungicide-treated than in undisturbed mesocosms. soil pathway, in which HLW is leaked from donor roots These unexpected results can be readily explained by the and/or associated mycorrhizal hyphae to soil, and is then fact that soil water potential in upper compartments was taken up by the roots and/or mycorrhizal fungal hyphae significantly lower in fungicide-treated than in undis- of the receiver seedlings. turbed mesocosms. Since soil water potential gradients Since the oak plants had the same size in both treat- provide the driving force for hydraulic redistribution ments, it remains to be explained why soil in upper (Caldwelletal.1998), a steeper potential gradient compartments was drier in fungicide-treated than in between taproot and upper compartments favored undisturbed mesocosms at the end of the experiment. HL in fungicide-treated mesocosms. As a result, One explanation would be that oaks in disturbed meso- HLW contributed a greater proportion of upper soil cosms may have maintained higher transpiration rates 70 Plant Soil (2012) 355:63–73 than those in undisturbed mesocosms, thus causing 1998;Augéetal.2001;Augé2004; Bearden 2001; faster depletion of soil water in upper compartments. Rillig 2004; Rillig and Mummey 2006). Fungal hyphae However, this explanation seems rather unlikely, are highly effective in stabilizing soil structure, as they because heavily ectomycorrhizal plants with abundant grow into the soil matrix to create a mesh that entangles extramatrical mycelium generally show greater water and holds soil particles together. Fungal hyphae and uptake ability, root hydraulic conductance, stomatal their exudates play a crucial role in the physical and conductance and transpiration rate than plants with chemical binding and stabilization of micro- and macro- lower mycorrhizal colonization (Duddridge et al. 1980; aggregates in soil (Miller and Jastrow 1990; Rillig and Guehletal.1992; Muhsin and Zwiazek 2002a, b; Mummey 2006;Wuetal.2008). The pore distribution Nardini et al. 2000; Morte et al. 2001;Bogeat-Triboulot and the moisture characteristics of a soil are strongly et al. 2004;Marjanovicetal.2005; Kennedy and Peay influenced by soil structure and aggregation level (Hillel 2007; Allen 2007). In particular, many studies have 1982), so that highly aggregated soils can hold more emphasized the important role that EMF extramatrical water than poorly aggregated soils. Interestingly, the hyphae play in the uptake and transfer of water to host amount of water that a soil can hold at relatively high plants (Brownlee et al. 1983; Duddridge et al. 1980; values of soil water potential (such as those in our study) Lamhamedi et al. 1992). Decreased water flow resis- is particularly strongly affected by soil structure (Hillel tance of the apoplast (Muhsin and Zwiazek 2002a)and/ 1982). Augé et al. (2001) found that a soil colonized by or increased water transport capacity of the plasma AMF hyphae lost more water than a non-mycorrhizal membrane of root cells (Marjanovic et al. 2005)often soil before its soil matric potential began to decline lead to enhanced root hydraulic conductance in ectomy- during a drying cycle. In other words, as the soil started corrrhizal plants. Arbuscular mycorrhizae also often to dry, slightly more water was available to plant roots in enhance water uptake, stomatal conductance and tran- mycorrhizal than in non-mycorrhizal soils at relatively spiration rates in their host plants (Allen et al. 1981; high soil matric potentials (Augé et al. 2001). Allen 1982; Allen and Boosalis 1983; Ruiz-Lozano and Whereas the influence of EMF extramatrical hyphal Azcón 1995; Ruiz-Lozano et al. 1995;Augé2001; density on soil moisture retention properties has 2004; Marulanda et al. 2003; Querejeta et al. 2006, received comparatively less attention, the effects of 2007b). Therefore, oaks in fungicide-treated mesocosms EMF (and saprotrophic) hyphae on soil aggregation would be expected to deplete soil water more slowly are similar to those of AMF hyphae (Emerson et al. than their heavily mycorrhizal (EM + AM) counterparts 1986; Tisdall et al. 1997; Caesar-TonThat and Cochran in undisturbed mesocosms, which is the opposite of 2000; Bogeat-Triboulot et al. 2004). Many EMF species what we found in our study. (including species of Scleroderma and Pisolithus)are A much more plausible explanation for the observed notoriously capable of producing extremely abundant differences in soil water potential between fungicide- extramatricalmycelium(Leakeetal.2004), so their treated and undisturbed mesocosms is that sharply re- potential influence on soil moisture retention properties duced hyphal density and viability may have affected could be even greater than that of AMF. In our study, the moisture retention properties of soil in the former differences in soil hyphal length density and viability treatment (Augé et al. 2001;Bearden2001;Rilligand between fungicide-treated and undisturbed mesocosms Mummey 2006). Decreased soil water holding capacity were actually much greater than those between non- due to reduced hyphal density in soil may have caused mycorrhizal and mycorrhizal systems in the study of lower water storage in upper compartments after irriga- Augé et al. (2001). Therefore an indirect fungicide effect tion, therefore leading to faster soil water depletion by on soil water holding capacity through a reduction of oaks in fungicide-treated compared to undisturbed mes- hyphal abundance and viability in the soil is both plau- ocosms. Also, soil water loss to evaporation could be sible and likely. The positive correlation found across higher when the buffering role of soil hyphae is absent, treatments between viable hyphal density and soil water particularly in a small soil volume. Numerous studies potential in upper compartments supports this interpre- have demonstrated that colonization of soil by arbuscu- tation of the data (R200.663; P00.026). This explana- lar mycorrhizal fungi (AMF) and concurrent changes in tion is also supported by the fact that donor oaks in extramatrical hyphal density can change soil moisture fungicide-treated mesocosms were using a greater retention properties (Schreiner et al. 1997; Jastrow et al. proportion of deuterium-labeled water from taproot Plant Soil (2012) 355:63–73 71 compartments than their undisturbed counterparts hyphal abundance and viability on hydraulic redistribu- at time of sampling (Fig. 2), which strongly sug- tion patterns is maintained across a wide range of soil gests decreased water availability in upper compart- moisture potential values. At broader scales, our results ments after fungicide application. Deuterium suggest that anthropic disturbances that alter the abun- enrichment of donor oaks was negatively related to dance and viability of fungal hyphae in soil (such as viable hyphal length (and, to a lesser extent, total application of fungicides or other soil contaminants) hyphal length), which also suggests decreasing soil might exert a significant impact on the hydrological water availability in upper root compartments (and functioning of natural and agricultural ecosystems heavier oak reliance on labeled water stored in taproot through changes in hydraulic redistribution patterns compartments) with decreasing soil hyphal density (Jackson et al. 2000). and viability. It is important to note that fungicide addition by itself did not alter the water holding capacity Acknowledgements This work was supported by the US of soil (see the Materials & Methods section), so National Science Foundation Biocomplexity Program (DEB 9981548), and by the Spanish Ministerio de Educación y Ciencia decreased hyphal density is the most plausible explana- (AGL2006-11234). Francisco M. Padilla made helpful comments tion for the lower soil water potentials in the upper root on an earlier draft of this manuscript. JI Querejeta acknowledges compartments of the fungicide-treated mesocosms. support from the “Ramón y Cajal” Program of the Spanish Root architecture was not assessed in this study, so Ministerio de Educación y Ciencia. The experiments reported here comply with the current laws of the country in which the experi- we cannot rule out the possibility that small differences ments were conducted (USA). in root length or density between treatments (due to disparities in fertilizer regime or mycorrhizal status) might have contributed to the observed differences in References upper soil water potential at time of sampling. However, this is implausible because fertilizer addition to non- Allen MF (1982) Influence of vesicular-arbuscular mycorrhizae inoculated mesocosms (later used as fungicide-treated on water movement through Bouteloua gracilis (H.B.K.) mesocosms) would be expected to lead to lower root: Lag ex Steud. New Phytol 91:191–196 shoot ratios and lower root growth and density (e.g., Allen MF (2007) Mycorrhizal fungi: highways for water and – Föhse et al. 1988; Berger and Glatzel 2001). Berger and nutrients in arid soils. 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