Sdarticle

Applied Soil Ecology 34 (2006) 276–279 www.elsevier.com/locate/apsoil

Short communication

Establishing arbuscular mycorrhiza-free soil: A comparison of six methods and their effects on nutrient mobilization Kerstin Endlweber *, Stefan Scheu Technische Universita¨t Darmstadt, Institut fu¨r Zoologie, Schnittspahnstr. 3, 64287 Darmstadt, Germany Received 22 October 2005; received in revised form 4 April 2006; accepted 4 April 2006

Abstract In order to study decomposer–mycorrhiza interactions, mycorrhiza-free treatments are often compared with reinoculated treatments. However, methods to achieve mycorrhiza-free soil, such as chloroform fumigation and autoclaving, cause strong side effects by increasing nutrient availability and changing microbial activity. We investigated the effectiveness of soil heating (60, 80, 100 and 120 8C), autoclaving and chloroform fumigation in eliminating arbuscular mycorrhizal (AM) fungi and also determined the impacts of the tested methods on mobilization of ammonium, nitrate and phosphorus as well as on microbial activity. Heating at 60 8C and chloroform fumigation reduced colonization of plant roots (Plantago lanceolata) by AM fungi to less than 1%, while all other treatments decreased colonisation to less than 0.2%. All six methods affected plant growth and plant tissue nutrient concentrations. This in part was due to mobilization of ammonium, nitrate and phosphorus in particular in autoclaved soil and soil heated at 100 and 120 8C. Heating soil at 60 8C also increased plant growth and shoot nutrient concentration, but had least side effect on nutrient mobilization and microbial activity, suggesting that moderate heating is preferable to other methods when setting up experiments investigating mycorrhiza–decomposer interactions. # 2006 Elsevier B.V. All rights reserved. Keywords: Mycorrhiza–decomposer interaction; Sterilization; Nutrient mobilization; Microbial activity

1. Introduction Arbuscular mycorrhizal (AM) fungi form symbiosis with about 80% of all terrestrial plant genera. By extending the absorptive surface of plant roots, AM fungi enable the plant to absorb relatively immobile ions, such as phosphate. In addition to phosphate and other nutrients, AM fungi transport inorganic nitrogen to plant roots (Javelle et al., 1999; Hawkins et al., 2000). Depending on environmental conditions the symbiosis of plants with AM fungi, usually considered as

* Corresponding author. Tel.: +49 6151 164299; fax: +49 6151 166111. E-mail address: [email protected] (K. Endlweber). 0929-1393/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2006.04.001

mutualistic, can shift from mutualistic to parasitic (Johnson et al., 1997). Mycorrhizal inoculation is reduced by fertilisation with phosphorus (Abott et al., 1984) and also at high nitrogen concentrations (Johnson et al., 2003; Blanke et al., 2005). However, symbiosis between plants and AM fungi is not only influenced by soil nutrient concentrations but also depends on the interaction with other soil organisms (Bakonyi et al., 2002; Tiunov and Scheu, 2005). To evaluate the effect of AM–decomposer interactions mycorrhiza-free soil is often compared with reinoculated soil. To achieve mycorrhiza-free treatments, the soil is often sterilised by gamma irradiation or autoclaved. Both methods are known to mobilize soil nutrients (N, P) and to change soil characteristics (Alphei and Scheu, 1993), changes which may influence

K. Endlweber, S. Scheu / Applied Soil Ecology 34 (2006) 276–279

the symbiosis between plants and mycorrhizal fungi. Consequently, the interpretation of plant responses, such as plant growth and shoot nutrient content, to mycorrhizal inoculation might be hampered. In the present study we evaluated the effectiveness, and side effects of, methods for eliminating AM fungi from soil. Soil was either heated (at 60, 80, 100 and 120 8C), autoclaved or fumigated with chloroform. We choose heat treatments, since we expected heating to cause only minor side effects compared to more conventional methods like autoclaving and chloroform fumigation. 2. Materials and methods The experiment was conducted in rhizotrons (height 20 cm, width 15 cm, thickness 1 cm) in a greenhouse (16 h light, 18 8C). Each rhizotron was filled with 100 g soil taken from the upper 20 cm of a set-aside field near Halle (Germany). Mycorrhiza spores and hyphae in soil were eliminated/reduced by six treatments. Soil (1 kg) was either heated at 60, 80, 100 and 120 8C for 4 h, autoclaved (120 8C, 2 h) or fumigated with chloroform (24 h). Heating time was restricted to 4 h to ensure uniform heating of soil and to avoid effects of strong dehydration. Each of the six treatments and control soil were replicated 8 times. Sub-samples for moisture determination as well as for measurement of nitrogen and phosphorus concentrations and microbial respiration were taken. Before analysis, sub-samples were stored at 15 8C for 1 week. Soil pH (H2O) was determined by a pH meter. Mineral nitrogen was extracted from sub-samples by addition of Alaun (KAI(SO4)2). Ammonium and nitrate concentrations were determined by distillation (Gerhardt Vadodest 20, Ko¨nigswinter, Germany). Ammonium concentration was determined by measuring colour change of an indicator (Mischindikator 5, Merck, Darmstadt, Germany) when titrated with sulphuric acid (H2SO4). Nitrate was reduced to ammonium by addition of Devarda reagent, distilled and titrated with sulphuric acid. Carbonate-extractable phosphate was extracted from soil by sodium hydrogen carbonate and measured photometrically. Under acidic conditions (pH 5) phosphate forms a blue complex reacting with ammonium molybdate, absorbing light at 880 nm. Soil respiration was measured at 22 8C using an automated respirometer based on electrolytic O2 microcompensation (Scheu, 1992). Surface sterilised seeds of Plantago lanceolata were sown in the rhizotrons. After germination, redundant seedlings were removed to leave one seedling per

277

rhizotron. The rhizotrons were watered every other day with 5 ml deionized water; plants were harvested after 5 weeks, and shoot dry weight was determined after drying at 60 8C for 3 days. The dried shoots were milled in a ball mill (Retsch, Haan, Germany) and plant N and C were determined by an elemental analyser (Carlo Erba, Milan, Italy). Roots were washed and cleared by boiling in 1N KOH. The roots were dyed in 1N HCl mixed with two drops ink (Quink, Parker Permanent Blue, Germany) overnight. After dying, roots were transferred into a mixture of lactic acid and distilled water and discoloured overnight. Colonisation of roots with VA mycorrhiza was estimated using the grid line intersect method (Giovannetti and Mosse, 1980). To determine root biomass, roots were dried at 60 8C for 3 days and weighed. Data were analysed by one-way ANOVA using SAS 6.12 (SAS Institute, Cary, N.C.). Differences between means were evaluated using Tukey’s honestly significant difference test. 3. Results 3.1. Soil nutrients and respiration Soil pH (average 7.14) was not significantly affected by the treatments. The ammonium concentration was significantly increased in the 100 8C, chloroform fumigation and autoclaved treatment compared to control, with a more than twofold increase in autoclaved soil compared to the control and the 60 8C heated soil (Fig. 1a). Soil nitrate concentration of the chloroform fumigated soil and the soil heated to 120 and 80 8C was lower than in the control. As for ammonium the nitrate concentrations were highest in autoclaved soil (Fig. 1b). Heating soil at 100 and 120 8C caused a more than twofold increase in carbonate-extractable phosphate compared to the control (Fig. 1c). Microbial activity was significantly increased when soil was heated at 120 8C and in autoclaved soil (Fig. 1d). 3.2. Plant performance Shoot biomass significantly exceeded that of control plants in the 100, 120 8C, autoclaved and chloroform fumigated treatments (Fig. 2a). Root biomass was significantly increased in the 100 8C and the chloroform fumigation treatments and was lowest in the control (Fig. 2b). Overall, biomass was highest in the 100 8C and chloroform fumigation treatments. Shoot carbon concentration followed that of shoot biomass. It increased from 40.5% in the control to an average of 43.1% in

278

K. Endlweber, S. Scheu / Applied Soil Ecology 34 (2006) 276–279

Fig. 1. Ammonium (a), nitrate (b) and phosphorus concentration (c) and microbial activity (oxygen consumption) (d) in heated soil (60, 80, 100 and 120 8C), autoclaved soil (aut), chloroform fumigated soil (chlor) and control soil (Ctr.). Bars sharing the same letter are not significantly different (Tukey’s honestly significant difference, P < 0.05).

heated/fumigated soil (Fig. 2c). Shoot nitrogen concentrations increased from 0.70% in the control and 1.72% in the 120 8C treatment to 1.85% in autoclaved soil (Fig. 2d). Consequently, shoot C/N ratio was highest in the control and the chloroform fumigation treatment.

In control plants 50% of the roots were colonised by mycorrhiza. In soil heated at 60 8C or fumigated with chloroform the infection rate was approximately 1%. In all other treatments colonisation rate was lower than 0.2 %.

Fig. 2. Shoot biomass (a), root biomass (b), shoot carbon (c) and shoot nitrogen in Plantago lanceolata grown in heated soil (60, 80, 100 and 120 8C), autoclaved soil (aut), chloroform fumigated soil (chlor) and control soil (Ctr.). Bars sharing the same letter are not significantly different (Tukey’s honestly significant difference, P < 0.05).

K. Endlweber, S. Scheu / Applied Soil Ecology 34 (2006) 276–279

4. Discussion Each of the soil treatments-heating, autoclaving or chloroform fumigation-reduced mycorrhizal inoculation rate. However, each treatment also exerted side effects and increased nutrient availability and plant growth. Both ammonium and nitrate concentrations were increased in heated and autoclaved soil, resulting in maximum shoot N-concentrations in autoclaved soil. Even though NO3 and NH4+ concentrations were only little affected by heating at 120 8C, plants grown in this soil had the second highest nitrogen concentrations. Thus, effects on plant nutrition and plant growth might have been due to factors other than nitrogen mobilization, or nitrogen pools in soil might have been mobilized more easily after heating. Indeed heating the soil at 120 8C strongly increased microbial activity, suggesting that micro-organisms mobilized nutrients after heating. Soil heating and chloroform fumigation reduce microbial biomass by killing part of the microbial population. Triggered by the increased availability of microbial carbon, microbial respiration rates increase (Serrasolsas and Khanna, 1995) and subsequently more nutrients might be mobilized. Therefore, increased carbon availability may further enhance plant growth. However, the effects of soil heating and fumigation on plant growth might also be due to modifications in the composition of the microbial community (Dickens and Anderson, 1999; Toyota et al., 1999). Each of the investigated soil heating and fumigation treatments affected plant growth and shoot nutrient concentrations, suggesting that side effects cannot be avoided entirely when eliminating mycorrhiza. When soil is reinoculated with mycorrhizal propagules high nutrient contents hamper root infection by mycorrhizal fungi (Azco´n et al., 1982; Blanke et al., 2005). Also, effects of mycorrhiza on shoot nitrogen concentrations and plant growth might be masked by nitrogen made available by soil heating and fumigation. This also hampers the investigation of interactions between mycorrhiza and decomposers since the effect of decomposers on plant growth may also be masked if nutrient concentrations in soil are high (Wurst et al., 2005). Overall, heating soil at 60 8C caused the least side effects and did not significantly affect soil nutrient concentrations. Nevertheless, plant biomass and shoot nutrient concentration were increased. However, heating soil to 60 8C also effectively reduced colonisation of roots by mycorrhizal fungi to less than 1%. Therefore, heating soil at 60 8C (and potentially less) is preferable to the other treatments studied for setting up experiments

279

investigating effects of mycorrhiza and their interaction with decomposer animals on plant growth. Acknowledgements We thank the UFZ Halle for providing us with the soil. Special thanks to Katja Rosenberg for her help during the experiment. References Abott, L.K., Robson, A.D., De Boer, G., 1984. The effect of phosphorus on the formation of hyphae in soil by the vesiculararbuscular mycorrhizal fungus, Glomus fasciculatum. New Phytol. 97, 437–446. Alphei, J., Scheu, S., 1993. Effects of biocidal treatments on biological and nutritional properties of a mull-structured woodland soil. Geoderma 15, 435–448. Azco´n, R., Gomez-Ortega, M., Barea, J.M., 1982. Comparative effects of foliar-or soil-applied nitrate on vesicular-arbuscular mycorrhizal infections in maize. New Phytol. 92, 553–559. Bakonyi, G., Posta, K., Kiss, I., Fabian, M., Nagy, P., Nosek, J.N., 2002. Density-dependent regulation of arbuscular mycorrhiza by Collembola. Soil Biol. Biochem. 34, 661–664. Blanke, V., Renker, C., Wagner, M., Fullner, K., Held, M., Kuhn, A.J., Buscot, F., 2005. Nitrogen supply affects arbuscular mycorrhizal colonization of Artemisia vulgaris in a phosphate-polluted field site. New Phytol. 166, 981–992. Dickens, H.E., Anderson, J.M., 1999. Manipulation of soil microbial community structure in bog and forest soils using chloroform fumigation. Soil Biol. Biochem. 31, 2049–2058. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 84, 489–500. Hawkins, H.J., Johansen, A., George, E., 2000. Uptake and transport of organic and inorganic nitrogen by arbuscular mycorrhizal fungi. Plant Soil 226, 275–285. Javelle, A., Chalot, M., Soderstrom, B., Botton, B., 1999. Ammonium and methylamine transport by the ectomycorrhizal fungus Paxillus involutes and ectomycorrhizas. FEMS Microbiol. Ecol. 30, 355– 366. Johnson, N.C., Graham, J.H., Smith, F.A., 1997. Functioning of mycorrhizal associations along the mutualism-parasitism continuum. New Phytol. 135, 575–586. Johnson, N.C., Rowland, D.L., Corkidi, L., Egerton-Warburton, L.M., Allen, E.B., 2003. Nitrogen enrichment alters mycorrhizal allocation at five mesic to semiarid grasslands. Ecology 84, 1895–1908. Scheu, S., 1992. Automated measurement of the respiratory response of soil microcompartments—active microbial biomass in earthworm feces. Soil Biol. Biochem. 24, 1113–1118. Serrasolsas, I., Khanna, P.K., 1995. Changes in heated and autoclaved forest soils of S.E. Australia. I. Carbon and nitrogen. Biogeochemistry 29, 3–24. Tiunov, A.V., Scheu, S., 2005. Arbuscular mycorrhiza and Collembola interact in affecting community composition of saprotrophic microfungi. Oecologia 142, 636–642. Toyota, K., Ritz, K., Kuninaga, S., Kimura, M., 1999. Impact of fumigation with metam sodium upon soil microbial community structure in two Japanese soils. Soil Sci. Plant Nutr. 45, 207–223. Wurst, S., Langel, R., Scheu, S., 2005. Do endogeic earthworms change plant competition? A microcosm study. Plant Soil 271, 123–130.