Applied Soil Ecology 34 (2006) 276–279

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Short communication
Establishing arbuscular mycorrhiza-free soil: A comparison of
six methods and their effects on nutrient mobilization
Kerstin Endlweber *, Stefan Scheu
Technische Universität Darmstadt, Institut für 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 mutualistic, can shift from mutualistic to parasitic

(Johnson et al., 1997).
Arbuscular mycorrhizal (AM) fungi form symbiosis Mycorrhizal inoculation is reduced by fertilisation
with about 80% of all terrestrial plant genera. By with phosphorus (Abott et al., 1984) and also at high
extending the absorptive surface of plant roots, AM nitrogen concentrations (Johnson et al., 2003; Blanke
fungi enable the plant to absorb relatively immobile et al., 2005). However, symbiosis between plants and
ions, such as phosphate. In addition to phosphate and AM fungi is not only influenced by soil nutrient
other nutrients, AM fungi transport inorganic nitrogen concentrations but also depends on the interaction with
to plant roots (Javelle et al., 1999; Hawkins et al., 2000). other soil organisms (Bakonyi et al., 2002; Tiunov and
Depending on environmental conditions the symbiosis Scheu, 2005).
of plants with AM fungi, usually considered as To evaluate the effect of AM–decomposer interac-
tions mycorrhiza-free soil is often compared with
reinoculated soil. To achieve mycorrhiza-free treat-
ments, the soil is often sterilised by gamma irradiation
* Corresponding author. Tel.: +49 6151 164299;
or autoclaved. Both methods are known to mobilize soil
fax: +49 6151 166111. nutrients (N, P) and to change soil characteristics
E-mail address: [email protected] (K. Endlweber). (Alphei and Scheu, 1993), changes which may influence

0929-1393/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2006.04.001
 K. Endlweber, S. Scheu / Applied Soil Ecology 34 (2006) 276–279 277

the symbiosis between plants and mycorrhizal fungi. rhizotron. The rhizotrons were watered every other day
Consequently, the interpretation of plant responses, with 5 ml deionized water; plants were harvested after 5
such as plant growth and shoot nutrient content, to weeks, and shoot dry weight was determined after
mycorrhizal inoculation might be hampered. drying at 60 8C for 3 days. The dried shoots were milled
In the present study we evaluated the effectiveness, in a ball mill (Retsch, Haan, Germany) and plant N and
and side effects of, methods for eliminating AM fungi C were determined by an elemental analyser (Carlo
from soil. Soil was either heated (at 60, 80, 100 and Erba, Milan, Italy). Roots were washed and cleared by
120 8C), autoclaved or fumigated with chloroform. We boiling in 1N KOH. The roots were dyed in 1N HCl
choose heat treatments, since we expected heating to mixed with two drops ink (Quink, Parker Permanent
cause only minor side effects compared to more Blue, Germany) overnight. After dying, roots were
conventional methods like autoclaving and chloroform transferred into a mixture of lactic acid and distilled
fumigation. water and discoloured overnight. Colonisation of roots
with VA mycorrhiza was estimated using the grid line
2. Materials and methods intersect method (Giovannetti and Mosse, 1980). To
determine root biomass, roots were dried at 60 8C for 3
The experiment was conducted in rhizotrons (height days and weighed.
20 cm, width 15 cm, thickness 1 cm) in a greenhouse Data were analysed by one-way ANOVA using SAS
(16 h light, 18 8C). Each rhizotron was filled with 100 g 6.12 (SAS Institute, Cary, N.C.). Differences between
soil taken from the upper 20 cm of a set-aside field near means were evaluated using Tukey’s honestly signifi-
Halle (Germany). Mycorrhiza spores and hyphae in soil cant difference test.
were eliminated/reduced by six treatments. Soil (1 kg)
was either heated at 60, 80, 100 and 120 8C for 4 h, 3. Results
autoclaved (120 8C, 2 h) or fumigated with chloroform
(24 h). Heating time was restricted to 4 h to ensure 3.1. Soil nutrients and respiration
uniform heating of soil and to avoid effects of strong
dehydration. Each of the six treatments and control soil Soil pH (average 7.14) was not significantly affected
were replicated 8 times. Sub-samples for moisture by the treatments. The ammonium concentration was
determination as well as for measurement of nitrogen significantly increased in the 100 8C, chloroform
and phosphorus concentrations and microbial respira- fumigation and autoclaved treatment compared to
tion were taken. Before analysis, sub-samples were control, with a more than twofold increase in autoclaved
stored at 15 8C for 1 week. Soil pH (H2O) was soil compared to the control and the 60 8C heated soil
determined by a pH meter. Mineral nitrogen was (Fig. 1a). Soil nitrate concentration of the chloroform
extracted from sub-samples by addition of Alaun fumigated soil and the soil heated to 120 and 80 8C was
(KAI(SO4)2). Ammonium and nitrate concentrations lower than in the control. As for ammonium the nitrate
were determined by distillation (Gerhardt Vadodest 20, concentrations were highest in autoclaved soil (Fig. 1b).
Königswinter, Germany). Ammonium concentration Heating soil at 100 and 120 8C caused a more than
was determined by measuring colour change of an twofold increase in carbonate-extractable phosphate
indicator (Mischindikator 5, Merck, Darmstadt, Ger- compared to the control (Fig. 1c). Microbial activity
many) when titrated with sulphuric acid (H2SO4). was significantly increased when soil was heated at
Nitrate was reduced to ammonium by addition of 120 8C and in autoclaved soil (Fig. 1d).
Devarda reagent, distilled and titrated with sulphuric
acid. Carbonate-extractable phosphate was extracted 3.2. Plant performance
from soil by sodium hydrogen carbonate and measured
photometrically. Under acidic conditions (pH 5) Shoot biomass significantly exceeded that of control
phosphate forms a blue complex reacting with plants in the 100, 120 8C, autoclaved and chloroform
ammonium molybdate, absorbing light at 880 nm. Soil fumigated treatments (Fig. 2a). Root biomass was
respiration was measured at 22 8C using an automated significantly increased in the 100 8C and the chloroform
respirometer based on electrolytic O2 microcompensa- fumigation treatments and was lowest in the control
tion (Scheu, 1992). (Fig. 2b). Overall, biomass was highest in the 100 8C and
Surface sterilised seeds of Plantago lanceolata were chloroform fumigation treatments. Shoot carbon con-
sown in the rhizotrons. After germination, redundant centration followed that of shoot biomass. It increased
seedlings were removed to leave one seedling per 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 concen- In control plants 50% of the roots were colonised
trations increased from 0.70% in the control and 1.72% in by mycorrhiza. In soil heated at 60 8C or fumigated
the 120 8C treatment to 1.85% in autoclaved soil with chloroform the infection rate was approximately
(Fig. 2d). Consequently, shoot C/N ratio was highest 1%. In all other treatments colonisation rate was
in the control and the chloroform fumigation treatment. 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) 276–279 279

4. Discussion investigating effects of mycorrhiza and their interaction

with decomposer animals on plant growth.
Each of the soil treatments-heating, autoclaving or
chloroform fumigation-reduced mycorrhizal inoculation Acknowledgements
rate. However, each treatment also exerted side effects
and increased nutrient availability and plant growth. Both We thank the UFZ Halle for providing us with the
ammonium and nitrate concentrations were increased in soil. Special thanks to Katja Rosenberg for her help
heated and autoclaved soil, resulting in maximum shoot during the experiment.
N-concentrations in autoclaved soil. Even though NO3
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