Ecological Engineering 57 (2013) 314–323

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Research paper

Soil aggregate stability related to soil density, root length, and

mycorrhiza using site-specific Alnus incana and Melanogaster
variegatus s.l.
Frank Graf a,∗ , Martin Frei b,1
a
WSL Institute for Snow and Avalanche Research SLF, CH-7260 Davos Dorf, Switzerland
b
Swiss Federal Institute for Forest, Snow and Landscape Research WSL, CH-8903 Birmensdorf, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Eco-engineering aims at stabilising soil and slopes by applying technical and biological measures. Engi-
Received 29 December 2012 neering structures are commonly well defined, immediately usable and operative, and their stability
Received in revised form 26 March 2013 effects quantifiable. Differently, the use of plants requires more restrictive boundary conditions and the
Accepted 6 April 2013
protection potential is rarely easily calculable and is developing as a function of growth time. Soil aggre-
Available online 17 May 2013
gation processes play a crucial role in re-establishing soil structure and function and, conclusively, for
successful and sustainable re-colonisation. Mycorrhizal fungi are key-players that foster the development
Keywords:
of a protective vegetation cover. They accelerate and increase plant growth and, additionally, contribute
Ectomycorrhiza
Alder
to soil aggregate stability which, on its part, was recently proposed as an appropriate indicator with regard
Root growth to the quantification of biological effects on soil and slope stability.
Soil mechanics The objective of this study was to determine the effects of mycorrhizal fungi on the host’s root system
as well as on soil aggregate stability. Furthermore, the biological contribution to soil aggregate stability
was compared to mechanical stabilisation effects due to soil compaction. The site-specific plant-fungus
symbiosis Alnus incana and Melanogaster variegatus s.l. of a recently stabilised steep catchment on moraine
was used for laboratory experiments.
Aggregate stability tests were performed with samples of differently treated moraine, including soil at
low (∼15.5 kN m−3 ) and high (∼19.0 kN m−3 ) dry unit weight, soil planted with A. incana (White Alder) as
well as the combination of planting with alder and inoculating with the mycorrhizal fungus M. variega-
tus s.l. After a 20 week growth period in a greenhouse, a total of 100 samples was tested and evaluated.
Positive correlations were found between the soil aggregate stability and the three variables dry unit
weight, root length per soil volume, and degree of mycorrhization. Based on robust statistics it turned out
that over all samples dry unit weight and degree of mycorrhization were strongest correlated with soil
aggregate stability. Simple linear regression models revealed a significant positive effect of root length
per soil volume on soil aggregate stability. Compared to the non-inoculated control plants, mycorrhized
White Alder produced significantly more roots and, consequently, higher soil aggregate stability. Further-
more, the combined biological effect of plant roots and mycorrhizal mycelia on aggregate stability in soil
with low density (∼15.5 kN m−3 ) was comparable to the compaction effect of the pure soil from 15.5 to
∼19.0 kN m−3 .
Literature data on the effect of vegetation on the angle of internal friction ˚’ of the same moraine
showed similar correlations, i.e. that ˚ of low density soil material (∼15.5 kN m−3 ) increased by the
same amount whether by planting with White Alder or by compaction to ∼19.0 kN m−3 . Based on this
coincidence and from a soil mechanical perspective, soil aggregate stability is suitable to estimate the
joint effect of plants and mycorrhizal fungi with respect to their contribution to soil and slope stability in
the near-surface layer.
© 2013 Elsevier B.V. All rights reserved.

∗ Corresponding author at: WSL Institute for Snow and Avalanche Research SLF, CH-7260 Davos Dorf, Switzerland. Tel.: +41 81 417 0210; fax: +41 81 417 0110.
E-mail addresses: [email protected] (F. Graf), [email protected] (M. Frei).
1
Current address: Office for Forest and Natural Hazards Grisons, Loëstrasse 14, CH-7000 Chur, Switzerland.

0925-8574/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ecoleng.2013.04.037
 F. Graf, M. Frei / Ecological Engineering 57 (2013) 314–323 315

1. Introduction 2000; Rillig et al., 2002; Ritz and Young, 2004; Bedini et al., 2009;
Martin et al., 2012). However, the contribution of ectomycorrhizal
A major goal of eco-engineering measures is their contribution fungi to the formation and stabilisation of soil aggregation has not
to slope stability. For that purpose, plant growth and the develop- been elaborated that comprehensively, yet. In spite of that, it has
ment of a functional vegetation cover are essential. However, on been hypothesised (Tisdall, 1991; Rillig and Mummey, 2006) and,
soil affected by erosion and sliding processes, even pioneer plants up to a certain extent, demonstrated that these fungi may play an
reach their limits (Coppin and Richards, 1990; Morgan and Rickson, important role in soil aggregation too (Thornton et al., 1956; Graf
1995; Gray and Sotir, 1996). and Gerber, 1997; Tisdall et al., 1997; Tagu et al., 2001; Graf et al.,
Depending on the degree of degradation of a soil, the pore struc- 2006; Ambriz et al., 2010).
ture is strongly destabilised due to the almost complete breakup of As a matter of fact, the filamentous growth-form and the vast
the soil aggregates. Hence, as water and nutrients are stored in the mycelial networks far beyond the rhizosphere make them perfectly
interstitial of stable soil aggregates, the build-up of a water and adapted in view of soil aggregation and stabilisation. Further-
nutrient reserve is strongly hampered as the main part is imme- more, the production of “sticky” metabolites (polysaccharides,
diately leached out. Under such extreme conditions a gap results hydrophobines) strongly supports a functional role in soil aggre-
between the basic demands of the plants and the effective sup- gate formation (Caesar-Ton That et al., 2001; Tagu et al., 2001;
ply at the time eco-engineering is applied. Consequently, the main Mankel et al., 2002).
requirement of eco-engineering and, actually, any soil restoration Evidence on the soil aggregation potential of ectomycorrhizal
practice should be to establish conditions that favour formation fungi was already given by Thornton et al. (1956) with their
of stable soil aggregates, thereby facilitating an important step in investigation on mycelial aggregation of sandy soil under Pinus
the creation of nutrient and water reserves as the basis of plant radiata. Tisdall et al. (1997) recorded significant higher aggregation
growth and vegetation establishment (Tisdall and Oades, 1982; (>50 ␮m) of a soil clay slurry (<2 ␮m) prepared from a Vertisol inoc-
Sollins et al., 1996). ulated with an ectomycorrhizal Hebeloma species compared to the
Conventional solutions to provide this basis are manifold. non-inoculated control. Similar findings are reported from Graf and
Among the most common are the use of synthetic soil condition- Gerber (1997). They analysed the aggregate stability of an artificial
ers to stabilise the soil surface (Scheffer and Schachtschabel, 1992) mixture composed of quartz sand, glass balls and peat of inoculated
and the application of fertiliser to overcome nutrient deficiency. (Laccaria bicolor, L. montana) and non-inoculated samples, main-
However, these “first aid” measures entail secondary effects, tained under sterile conditions. The two inoculated treatments
notably fertilising (Cernusca, 1986; Arnolds, 1989, 1992a,b). The significantly increased the aggregate stability compared to the con-
pronounced growth stimulation of the above ground biomass com- trol and there was a considerable but non-significant difference
pared to roots and the preference of plants that are nutrient between the two Laccaria species.
indigent in place of autochthonous species are disadvantageous The species specificity of ectomycorrhizal fungi in their poten-
in the long-term. Furthermore, fertilisers hamper the develop- tial of soil aggregate formation and stabilisation was shown by Graf
ment of important micro-organisms, particularly mycorrhizal fungi et al. (2006). In their study, the fine fraction (<2 mm) of moraine
(Wallenda and Kottke, 1998). These essential plant partners are was inoculated with different ectomycorrhizal species (Hebeloma
already drastically decreased, in dependence of the magnitude of alpinum, Hebeloma repandum, Inocybe lacera, L. bicolor (2 stems), L.
the erosive and sliding incidents (Biondini et al., 1985; Biondini montana) and tested against the non-inoculated control. Except for
and Redente, 1986; Amaranthus and Trappe, 1993) as well as in I. lacera and stem 1 of L. bicolor, all other ectomycorrhizal fungi sig-
relation to the time the corresponding plant partners are missing nificantly increased soil aggregate stability. Laccaria bicolor (stem
(Parke et al., 1984). 2) and H. alpinum were the most effective soil aggregate stabilisers,
Recent research and experience suggest that the introduction followed by L. montana and H. repandum.
of indigenous plant species together with a managed community Similar findings for arbuscular mycorrhizal fungi were reported
of mycorrhizal fungi is an excellent approach to initiate and pro- from Schreiner et al. (1997) who found that Glomus mosseae
mote autogenic recovery of degraded ecosystems (Requena et al., improved the stabilisation of the aggregate class 2–4 mm signifi-
2001; Caravaca et al., 2003; King and Hobbs, 2006; Chaudhary et al., cantly more than Glomus etunicatum and Glomus rosea. Moreover, it
2009). Particularly in harsh, limiting environments, this procedural was demonstrated that different isolates of the same fungal species
method bears the potential for a substantial increase in long-term (Glomus intraradices) may differ in their efficiency of soil aggregate
revegetation success, by creating synergies between abiotic and formation and stabilisation (Bedini et al., 2009).
biotic processes (Jeffries et al., 2003; Byers et al., 2006). Indirectly, the mycorrhizal fungi affect the soil aggregate sta-
It is well known that most plants and nearly all species used bility through their host plants, particularly by accelerating the
for eco-engineering measures live in a symbiotic relationship with development of their root network and by serving as a distribution
mycorrhizal fungi. Previous studies have shown that mycorrhi- vector for associated micro-organisms, mainly bacteria and archaea
zal fungi improve plant growth by increasing the above ground themselves soil stabilising alike (Budi et al., 1999; Filion et al., 1999;
biomass as well as the root network (Cairnay and Chambers, 1999; Bezzate et al., 2000; Hildebrandt et al., 2002; Mansfeld-Giese et al.,
Smith and Read, 2008). Furthermore, these fungal partners protect 2002). Unlike mycorrhizal fungi that strongly influence at the scale
their host plants against soil borne pathogens and toxic elements of macro-aggregates, bacteria and archaea more directly partici-
and compounds potentially present in the soil solution (Kottke pate in the formation and stabilisation of micro-aggregates (Chenu,
et al., 1998; Brunner, 2001). In view of plant communities, it was 1989; Oades, 1993).
found, that mycorrhizal fungi influence plant diversity, ecosystem With all due respect to these important fungal traits, it should
productivity and, hence, regulate or accelerate succession pro- not be forgotten that the selection of appropriate species needs to
cesses (van der Heijden et al., 1998). experience careful examination. Sound information on their ecol-
Another important issue is their contribution to the formation of ogy and sociology is required depending on the plant species used
the soil structure and its strength at different spatial scales directly as initial step in the re-colonisation process as well as in view of
by charge, adhesive, and enmeshment mechanisms. These interre- the climax association in mind. Commercially available inoculum
lationships have been demonstrated time and again for arbuscular does not always fit the site-specific requirements and long-term
mycorrhizal fungi (Tisdall and Oades, 1982; Miller and Jastrow, perspectives. It is well known that not every mycorrhizal fungus
316 F. Graf, M. Frei / Ecological Engineering 57 (2013) 314–323

forms mycorrhiza with every plant. Additionally, there are suc- 2.2. Sample preparation
cessional processes in mycorrhizal communities in the way that
perennial plants do have other fungal partners in their juvenile, With regard to the performance of the aggregate stability tests
prime, and senescent living phase, respectively (Last et al., 1983; the soil material was moistened to a water content of 6% and
Graf, 1994). Consequently, prospects of success and efficiency are tamped into PVC-plastic tubes (diameter: 70 mm; height: 140 mm)
greatest if site-specific and compatible plants and mycorrhizal aiming for an average dry unit weight of ∼19 kN m−3 for the
fungi are selected. compacted soil samples (= compacted) and of ∼15 kN m−3 for the
In order to consider these framework conditions we used the loose soil samples (= soil), respectively. Four different treatments
soil material (moraine) of a recent landslide area, autochthonous were applied including untreated soil samples at the two differ-
White Alder (A. incana) and isolated one of the mycorrhizal part- ent dry unit weights as well as loose soil samples (∼15 kN m−3 )
ners (Melanogaster variegatus s.l.) forming symbiosis during the planted with Alnus incana (L.) Moench only (=planted), as well as
juvenile phase of its host plant. With these ingredients, differ- additionally inoculated with the mycorrhizal fungus Melanogaster
ently treated soil samples were prepared. Besides the untreated soil variegatus s.l. (=mycorrhized).
at low (∼15.5 kN m−3 ) and high dry unit weight (∼19.0 kN m−3 ), The inoculation was performed with mycelium – derived from
soil planted with A. incana as well as soil planted with A. naturally mycorrhized root tips of A. incana – of cultures on mod-
incana and inoculated with the mycorrhizal fungus M. variega- ified Melin Norkrans (MMN) agar (Marx and Bryan, 1975). The
tus s.l. were analysed; the latter two treatments at low dry unit amount of one fourth of a Petri dish was applied to each sample,
weight (∼15.5 kN m−3 ). The soil aggregate stability was analysed previously cut into small cubes of about 4 mm3 under sterile con-
and calculated according to Frei et al. (2003) and Burri et al. ditions. For the planted and mycorrhized treatments 15 alder seeds
(2009). Additionally, the root length per soil volume was mea- were applied to each sample and reduced to three seedlings after
sured to compare between inoculated and non-inoculated alder four weeks of growing. The samples were arranged completely ran-
roots. domly and maintained in a greenhouse for 20 weeks with 16 h of
The main hypotheses were that the inoculation with a natu- daylight and a temperature of 17 ◦ C (day) and 10 ◦ C (night), respec-
rally occurring mycorrhizal fungus of A. incana (i) increases the tively. Totally, 100 samples were subject of aggregate stability tests
root growth of the host plant and (ii) increases the soil aggregate consisting of 18 compacted, 34 soil, 29 planted and 19 mycorrhized
stability and (iii) that the increase in soil aggregate stability due specimens.
to mycorrhized plants is comparable to the stability of untreated
moraine with increased dry unit weight. Furthermore, the results
are compared to effects of roots on the angle of internal fric- 2.3. Fungal inoculum
tion ˚ addressed in a preceding investigation using site-specific
White Alder and moraine of the same investigation area (Graf et al., The inoculum applied was originally isolated from mycor-
2009). rhized roots of A. incana plantlets used in trap cultures with
the soil material under investigation. The identification of the
fungus was performed with a molecular-genetic analysis accord-
2. Material and methods ing to Peter (2000). ITS1 and ITS4 sequences were analysed at
the laboratories of the Mycosynth AG (Balgach, Switzerland). A
2.1. Investigation area and soil material nucleotide query was conducted with the database BLAST (2012,
www.ncbi.nlm.nih.gov/blast/Blast.cgi) to find similarities between
The soil investigated is a moraine of the subalpine landslide area sequences and to check for the most probable species.
“Schwandrübi” at Dallenwil-Wirzweli in the Canton of Nidwalden
in Central Switzerland. The “Schwandrübi” is a steep amphitheatre-
like gully on moraine and a part of a larger catchment (Burri et al., 2.4. Aggregate stability experiments and root processing
2009). Until 1980 it was a steady source of bed load of the outlet
channel and, therefore, a potential danger of the ski resort Wirzweli The determination of the water stability of aggregates was car-
above as well as the subjacent village Dallenwil. During 1981 and ried out according to a slightly modified protocol of Burri et al.
1982 joint technical and biological measures, in particular gabions, (2009) and Frei (2009). A sieve with mesh openings of 20 mm was
log cribwalls, the application of Salix purpurea cuttings and planting placed in a Plexiglas pot. After the greenhouse period, each sam-
of Alnus incana had been taken on a large scale. ple was put singly on the sieve. Subsequently, the pot was filled
The subsequently described experiments were performed with with water entirely covering the sample. After 5 min, the water was
untreated soil, i.e. bare moraine without vegetation cover, repre- drained. In a next step, the roots of planted as well as of planted
senting the situation at the time of the stabilisation and restoration and inoculated samples were carefully removed. Then, indepen-
measures in 1981. The corresponding soil material was analysed dent of treatment, the soil portion remaining on the sieve and the
physically and chemically. For a proper soil classification from a one passing (components ≤20 mm) were separately oven dried
geotechnical point of view (ASTM D 2487, 2002) the grain size for 24 h at 105 ◦ C. The soil aggregate stability was defined as the
distribution was performed according to ASTM D 422 (2000), dry weight ratio between the components above the sieve (aggre-
including the liquid limit and the plasticity index (ASTM D 4318, gates > 20 mm) and the sum of all components, above and below
2000). Furthermore, proctor standard compaction tests were con- the sieve. The soil portion remaining on the sieve consisted exclu-
ducted to get the maximum dry unit weight at optimum water sively of aggregates, as the maximum grain size of the soil samples
content (ASTM D 698, 2000). From oven dried material (24 h at was 10 mm. The portion passing the sieve was a mixture of single
105 ◦ C) the fractions ≤10 mm were used to prepare the samples for grains and aggregates equal to or smaller than 20 mm.
conducting all aggregate stability tests. The roots were cleaned, spread out in a water-filled transpar-
Chemical analysis included the quantification of exchangeable ent plastic container, and analysed with a flat-bed scanner. The
cations (Ca2+ , Mg2+ , K+ , Na+ ), determination of organic matter by total root length was determined using the software WinRhizo
oxidation with H2 O2 , and soil pH measurement in 0.01 M CaCl2 ® (2000). The root length per sample volume (cm cm−3 ) was used
(Schlichting et al., 1995; Brunner et al., 2002). as an indicator for plant growth. The degree of mycorrhization
 F. Graf, M. Frei / Ecological Engineering 57 (2013) 314–323 317

clay silt sand gravel Table 1

100 Cation exchange capacity of the moraine (GC-CL) from the investigation area
“Schwandrübi” in Dallenwil-Wirzweli (Switzerland).
90
mval kg−1 %
percent passing [%]

80
2+
70 max. particle size: 10 mm Ca 132.70 93.97
max. particle size: 63 mm Mg2+ 6.97 4.94
60 K+ 1.04 0.74
50 Na+ 0.50 0.35

40 Total 141.17 100.00

30

20 The pH[CaCl] of the soil material was 7.7 and the organic matter
10 content 0.2 ± 0.1% by weight. The total cation exchange capacity
amounted to 141.17 mval kg−1 . The corresponding contributions
0
0.001 0.01 0.1 1 10 100 of the individual ions (Ca2+ , Mg2+ , K+ , Na+ ) are listed in Table 1.
particle size [mm]
3.2. Fungal inoculum
Fig. 1. Grain size distribution curves of the soil material. : distribution curve of
the moraine including the coarse grains up to 63 mm, •: distribution curve of the soil The comparison of the DNA (ITS2, ITS4) with a reference data
material including grain sizes smaller than 10 mm used for all aggregate stability base of BLAST (www.ncbi.nlm.nih.gov/blast/Blast.cgi) assigned the
tests in this study as well as for the triaxial compression tests in the study of Graf
species under investigation to the genera Melanogaster (Melanogas-
et al. (2009).
traceae). With the molecular data, no further differentiation was
possible on species level between Melanogaster broomeanus Berk.
was determined under a stereo microscope (Wild M8) applying apud Tul. & C. Tul, M. variegatus (Vittad.) Tul. & C. Tul., and M. vitta-
the gridline intersection method after Brundrett et al. (1996). dinii Soehner et Knapp. The latter achieved a slightly higher score in
the ITS2 but a somewhat lower one in the ITS4 sequence compared
to the former two.
2.5. Statistical analysis Considering data on ecology, geography, and host species as well
as recent taxonomic discussions (Montecchi and Sarasini, 2000), M.
All statistical calculations were performed with the software R broomeanus and M. variegatus are the ones to be considered. Until
2.15.1 (R Development Core Team, 2012). Differences in aggregate further taxonomic clarification we follow a wider species concept
stability, dry unit weight, rooting, and among the four treatments and, based on the genus revision of Tulasne and Tulasne (1851),
of the 100 successfully maintained samples were calculated with refer to our species as M. variegatus s.l. for the time being.
robust statistics. Kruskal–Wallis and pair-wise Wilcoxon rank sum
tests were applied considering p-value adjustment for multiple
3.3. Correlations and sample characteristics
testing (Abdi, 2007). Furthermore, the Pearson and Spearman cor-
relation between aggregate stability, dry unit weight, rooting, and
The 100 samples tested and evaluated were composed of 34
mycorrhization degree were calculated under consideration of all
untreated soil samples (= soil), 29 samples planted with A. incana
samples (Stahel, 2000).
(= planted), 19 samples planted with A. incana and inoculated with
Due to unbalanced design, a weighting factor (ω) was applied
M. variegatus s.l. (= mycorrhized), and 18 samples of untreated
following the formula: t compacted soil (= compacted). The highest correlation among the
ωi = (N/ni t) with the total number of specimens (N = n)
i i considered variables was found between root length per soil vol-
divided by the total number of specimens per treatment (ni , i = 1,
ume and mycorrhization degree (0.86). The soil aggregate stability
2,.., t) and the number of treatments (t).
was noticeably correlated with the dry unit weight (0.63) as well
In order to test the effect of root length on soil aggregate sta-
as with the mycorrhization degree (0.51). A complete survey on
bility, robust simple linear regression models were fitted using an
Spearman and Pearson correlation is given in Table 2.
MM estimator (Koller and Stahel, 2011). First aid transformations
√ After the 20 week growth period in the greenhouse the dry
after Tukey (1957) were applied to the response (asin y) and the
unit weight was about 19 kN m−3 for the compacted samples
explanatory variable (log10 ).
and around 15.5 kN m−3 for the other three treatments (Tabs.
Residual analysis was conducted to check the compliance of the
3, 4). The Kruskal–Wallis rank sum test confirmed a significant
assumptions required and the fit of the models. For that purpose
treatment effect on the dry unit weight (X2 = 1248.7, df = 3, p-
residuals against fitted values (Tukey-Anscombe plot) and against
value = 2.2 × 10−16 ). According to the pairwise Wilcoxon rank sum
leverages (hat matrix) were analysed as well as the residual distri-
test the dry unit weight of the compacted samples was sig-
bution and the quantil–quantil plot.
nificantly higher compared to the other three treatments with

3. Results Table 2
Pearson (upper half of the matrix) and Spearman correlation (lower half of the
3.1. Soil analysis matrix) of soil aggregate stability [0,1], dry unit weight  [kN m−3 ], root length per
soil volume [cm cm−3 ], and mycorrhization degree [0,1].

The grain size distribution including the coarse parts up to Aggregation Dry unit Root Mycorrhization
63 mm is divided into: 75.3% gravel, 15.5% sand, 7.1% silt and 2.1% weight length
clay (Fig. 1). With a liquid limit of 21.5% and a plasticity index of Aggregation 1.00 0.63 0.38 0.51
8.6% it was classified as clayey gravel with sand (GC-CL). At an opti- Dry unit weight 0.57 1.00 0.28 0.16
mum water content of 7.9% the maximum dry unit weight was Root length 0.31 0.09 1.00 0.86
Mycorrhization 0.48 0.02 0.68 1.00
21.9 kN m−3 . The porosity constituted 0.467 m3 m−3 .
318 F. Graf, M. Frei / Ecological Engineering 57 (2013) 314–323

amount of stable aggregates # > 20 mm [0,1]

20
0.8
dry unit weight γ [kN m−3]

19 0.7
0.6
18
0.5

17 0.4
0.3
16
0.2
0.1
15
0
soil planted mycorrhized compacted soil planted mycorrhized compacted

Fig. 2. The dry unit weight  [kN m−3 ] of the samples after the 20 week growth Fig. 3. The aggregate stability in dependence of the four different treatments
period in the greenhouse in dependence of the four different treatments applied applied (soil, planted, mycorrhized, compacted), measured as ratio between the
(soil, planted, mycorrhized, compacted). dry weight of the stable fraction of particles bigger than 20 mm and the dry weight
of the whole sample.

p-values < 0.001. Among these latter three treatments (soil,

planted, mycorrhized) no significant differences were noticed median absolute deviations is given in Tables 3 and 4, respectively.
(Table 5; Fig. 2). The treatment effect on the soil aggregate stability was signifi-
cant (Kruskal–Wallis: X2 = 1538.7, df = 3, p-value = 2.2 × 10−16 ). The
3.4. Soil aggregate stability and root development pairwise Wilcoxon rank sum test revealed, on the one hand, no
significant difference between the treatments mycorrhized and
Whereas the mean aggregate stability values of the treatments compacted (p-value = 0.176). On the other hand, it turned out that
soil (0.03) and planted (0.06) were higher than the corresponding the latter two significantly differed from both soil and planted in
medians (0.01, 0.03) it was the other way round for the treat- all possible combinations with p-values <0.001 (Table 5; Fig. 3).
ments mycorrhized (mean: 0.38, median: 0.41) and compacted After the 20 week growth period a significant difference
(mean: 0.48, median: 0.62). A complete survey on the mean val- in root length per soil volume [cm cm−3 ] was found between
ues and corresponding standard deviations as well as medians and the inoculated (=mycorrhized) and non-inoculated (=planted)

Table 3 Table 4
Mean and standard deviation (sd) of soil aggregate stability [0,1] and dry unit Median and median absolute deviation (mad) of soil aggregate stability [0,1] and dry
weight  [kN m−3 ] in terms of the four different treatments (A = soil, B = planted, unit weight  [kN m−3 ] in terms of the four different treatments (A = soil, B = planted,
C = mycorrhized, D = compacted), and of root length per soil volume [cm cm−3 ] in C = mycorrhized, D = compacted), and of root length per soil volume [cm cm−3 ] in
terms of the treatments B and C. terms of the treatments B and C.

Aggregate stability Dry unit weight Root length Aggregate stability Dry unit weight Root length

Mean sd Mean sd Mean sd Median Mad Median Mad Median Mad

Soil 0.032 0.041 15.2 0.309 0 0 Soil 0.010 0.015 15.2 0.297 0 0
Planted 0.059 0.065 15.3 0.436 0.62 0.196 Planted 0.030 0.030 15.5 0.445 0.59 0.104
Mycorrhized 0.380 0.212 15.5 0.415 1.50 0.758 Mycorrhized 0.410 0.267 15.6 0.297 1.52 1.127
Compacted 0.476 0.263 19.2 0.489 0 0 Compacted 0.615 0.163 19.3 0.667 0 0

Table 5
Kruskal–Wallis and pairwise Wilcox tests of (i) dry unit weight  [kN m−3 ] and (ii) soil aggregate stability [0,1] of the four different treatments (A = soil, B = planted,
C = mycorrhized, D = compacted), as well as Wilcox test of (iii) root length per soil volume [cm cm−3 ] of the treatments B and C (bold numbers: significant p-values).

(i)
Dry unit weight ∼ treatment (A, B, C, D): X2 = 1248.7, p-value < 2.2 × 10−16

Soil Planted Mycorrhized

Planted 1.00
Mycorrhized 0.052 1.00
Compacted 2.3 × 10−8 6.7 × 10−8 × 10−6
1.2

(ii)
Aggregate stability ∼ treatment (A, B, C, D): X2 = 1538.7, p-value < 2.2 × 10−16

Soil Planted Mycorrhized

Planted 0.025
Mycorrhized 1.2 × 10−8 1.7 × 10−7
Compacted 1.1 × 10−7 6.5 × 10−7 0.176

(iii)
Root length per soil volume ∼ treatment (B,C): W = 57.5, p-value < 4.5 × 10−6 .
 F. Graf, M. Frei / Ecological Engineering 57 (2013) 314–323 319

root length per soil volume [cm cm−3] demands, however, certain restrictive requirements in respect of
soil structural stability and, concomitant, water and nutrient sup-
2.5 ply (Graf, 1997; Graf et al., 2006). The formation of soil aggregates
is a key process with regard to these essential requirements and
it is widely accepted that stable micro- and macro-aggregates are
2.0
interactively assembled by micro-organisms (bacteria, fungi) and
plant roots (Rillig and Mummey, 2006).
1.5 The results of the present investigation dovetail with this con-
cept showing significant increases in soil aggregate stability from
pure soil samples without roots to the planted samples with
1.0 a root length of 0.6 cm cm−3 to the samples with mycorrhized
roots (1.5 cm cm−3 ). Furthermore, a high positive linear correla-
tion is obvious between soil aggregate stability and root length
0.5 per soil volume for non-mycorrihzed and, even more pronounced,
for mycorrihzed roots (Fig. 5). The difference in aggregate stabil-
planted mycorrhized ity between pure and planted soil can be attributed to stabilising
effects of the roots, particularly mechanical armouring. It is, how-
Fig. 4. The rooting performance of Alnus incana plantlets after the 20 week ever, more difficult to untangle the considerable increase in stable
growth period in the greenhouse measured as the root length per soil volume [cm aggregates from the planted to the mycorrhized samples. It seems
cm−3 ]: 29 non-inoculated individuals (planted, dark grey) and 19 inoculated with obvious that the mycorrhizal fungus, on the one hand, stimulated
Melanogaster variegatus s.l. (mycorrhized, light grey). root growth (Figs. 4 and 5). Such phenomenon has been recorded
for many host plants of both arbuscular and ectomycorrhiza (Smith
samples (Fig. 4). The average value of mycorrhized roots was and Read, 2008). Within this study this is supported by a correlation
1.5 (±0.76) cm cm−3 compared to 0.62 (±0.20) cm cm−3 for non- coefficient of 0.86 between root length and mycorrhization degree
mycorrhized roots (Tables 3 and 4). According to the Wilcox and the fact that alder plantlets inoculated with Melanogaster varie-
rank sum test this difference was significant (W = 57.5, p- gatus s.l. produced more than twice the root length per soil volume
value = 4.5 × 10−6 ). The mean value of the mycorrhization degree compared to the non-inoculated specimen. On the other hand it is
and the corresponding standard deviation were 46.1% ± 24.0. quite likely that the fungus directly increased soil aggregate stabil-
The simple linear regression models for soil aggregate stabil- ity.
ity as a function of root length per soil volume of the treatments According to Rillig and Mummey (2006) the direct effects of fun-
planted, mycorrhized, and the combination of them revealed sig- gal mycelia on soil aggregate stability can be loosely categorised
nificant positive root effects with p-values <0.001 (Fig. 5). The into biochemical, biophysical, and biological processes. Biochemi-
respective Spearman correlation was 0.57 (planted), 0.86 (mycor- cally and with respect to the ectomycorrhizal species used in this
rhized), and 0.83 (combination). study, mucilages, polysaccharides, and other extracellular com-
pounds may have contributed to soil aggregate stability (Chenu,
4. Discussion 1989; Caesar-Ton That et al., 2001; Bogeat-Triboulot et al., 2004;
Ambriz et al., 2010) as well as hydrophobins and related proteins
Eco-engineering methods generally aim at contributing to
which are supposed having a functional role in soil aggregation
slope stability. The successful application of biological measures
(Tagu et al., 2001; Mankel et al., 2002; Linder et al., 2005).
Another stabilising process is the alignment of primary particles,
such as clay. Growing hyphae are able to exert pressure on parti-
cles of their nearest neighbourhood forcing clay and organic matter
non−myc. roots (R² = 0.57)
1.0 together (Tisdall, 1991; Chenu and Stotzky, 2002). Under physio-
mycorrhized roots (R² = 0.86)
stable aggregates # > 20mm [asin( y )]

both (R² = 0.83) logical (or neutral) pH values, micro-organisms have a net negative
charge and mostly amino- and carboxyl groups on their surface
0.8 (Marshall, 1988). In a study on fungal bioweathering (Burford et al.,
2003) it was found that soil aggregates stabilised by extracellu-
lar polysaccharides persisted for longer when polyvalent cations,
0.6 e.g. Al3+ or Ca2+ were present, suggesting that negatively charged
polysaccharides were bound to negatively charged clay particles by
bridges of polyvalent cations (Martin, 1971). Likewise, such bridges
0.4 are formed between fungal hyphae and particles of clay having
proven to be stronger than some direct bonds between clay and
organic matter (Tisdall, 1991).
0.2 About 99% of the total cation exchange capacity of the moraine
used in this study was made up by the polyvalent cations Ca2+ and
Mg2+ . Therefore, as all negative charges on clay minerals are neu-
0.0 tralised, only a thin layer of positive charge surrounds the clay
particles. Irrespective of equal charges, this thin layer may have
−0.4 −0.2 0.0 0.2 0.4 enabled particles to come close together for attractive Van der
root length per soil volume [log10(cm cm−3)] Waals’ forces to take hold and for the clay to flocculate which fur-
√ ther supported the soil aggregation process. Although this process
Fig. 5. Simple linear regression models of soil aggregate stability [asin( y)] as a
proceeds independently of micro-organisms, the fungal mycelial
function of root length per soil volume [log10 (cm cm−3 )] of non-mycorrhized (dark
grey), mycorrhized roots (light grey) and the combination of them (black dash-dot network multiplies the effect, forms stronger bonds, and over-
line). comes spatial limitations (Tisdall, 1991; Rillig and Mummey, 2006).
320 F. Graf, M. Frei / Ecological Engineering 57 (2013) 314–323

Table 6
Comparison of aggregate stability (A: this study) and angle of internal friction˚ (B: Graf et al., 2009) as well as dry unit weight  and root length per soil volume from both
studies (A, B). All experiments were performed with the same soil (moraine) and plant material (Alnus incana); n.a.: not available.

Soil Planted Mycorrhized Compacted

A B A B A A B

Dry unit weight [kN m−3 ] 15.2 15.7 15.3 15.3 15.5 19.2 18.9
Root length [cm cm−3 ] 0 0 0.6 1.3 1.5 0 0
Aggregate stability [0,1] 0.03 n.a. 0.06 n.a. 0.38 0.48 n.a.
Angle of internal friction [◦ ] n.a. 34.3 n.a. 39.4 n.a. n.a. 40.1

The important role of fungal and clay interactions in the processes relevant soil classification (GC-CL in this study). Both aspects are
of soil evolution has been shown amongst others by the investiga- reasonably well accommodated by the specimen size used in this
tion of Tisdall et al. (1997) on soil clay aggregation by saprophytic study (height: 140 mm; diameter: 70 mm) which is a standard for
(Rhizoctonia solani and Hyalodendron sp.) and mycorrhizal fungi triaxial compression tests, allowing a maximum grain size <1/5 of
(Hymenoscyphus ericae and Hebeloma sp.). the specimen diameter (DIN18137-2, 2011; Arnold et al., 2005).
From a biophysical point of view, hyphae enmesh and entangle Due to these adaptations of sample size and aggregate dimen-
small inorganic and organic soil particles in the manner of roots sion of peculiar interest, the results may differ from other studies
but on a smaller scale (Tisdall and Oades, 1982; Miller and Jastrow, using “small-scaled approaches” (Le Bissonnais et al., 2007). How-
2000). Interdependently, roots and mycorrhizal hyphae contribute ever, with regard to slope stability and, in particular, to the
to the stability of micro- and macro-aggregates at different scales quantification of biological effects on a large scale, the method used
and, consequently, to the stability of the soil structure, i.e. the in this study is supposed to be more reliable. Additionally, it has the
soil matrix and its corresponding void space. Ambriz et al. (2010) advantage of comparability to standardised soil mechanical test
reported positive and significant correlation between root volume procedures.
of Fraxinus uhdei and water stable aggregates for plants inoculated Whereas the key role of soil aggregate stability in ecosystem
with the arbuscular fungus Glomus intraradices for aggregates of functioning is well known concerning water, gas, and nutrient
0.25 mm and inoculated with the ectomycorrhizal species Pisolithus fluxes (Angers and Caron, 1998; Amezketa, 1999; Eldridge and Leys,
tinctorius for aggregates of 1.0 and 0.5 mm. Bedini et al. (2009) 2003; Wick et al., 2009), only limited information is available with
found significantly higher amounts of stable aggregates of 1–2 mm regard to soil mechanical and geotechnical aspects. Although, some
in mycorrhizal compared to non-mycorrhizal soil which was fur- investigations on the effectiveness of soil aggregate stability in
ther positively correlated with mycorrhizal root volume as well view of protecting against erosion processes have been performed
as with the total hyphal length and hyphal density of arbuscular (Barthès and Roose, 2002; Barni et al., 2007; Canton et al., 2009;
mycorrhizal fungi. According to Bearden and Petersen (2000) and Graf et al., 2009; Pohl et al., 2009, 2012) almost nothing is known
Bearden (2001) the formation of aggregates in the class 1–2 mm in terms of slope stabilisation to preventing superficial landslides.
is associated with hyphal length of the mycorrhizal fungi and not In order to confer the effects of plants and mycorrhizal fungi
with root growth. However, both parameters are involved in the on soil aggregation to parameters relevant to slope stability
formation of aggregates >2 mm. It is not possible to fractionate the assessment, it is indispensable to correlate them somehow to con-
contribution of roots and fungal hyphae for the present study, due ventional soil failure concepts. From a simplified soil mechanical
to the fact that hyphal length was not addressed. However, the con- perspective the angle of internal friction (˚’) characterises the
siderable correlation (0.51) between soil aggregate stability and stability of a cohesion-less soil as was the moraine used in this
mycorrhization degree seems to support the direct effect of the investigation (Terzaghi and Peck, 1967).
fungus. Recently, a new approach has been proposed assigning biologi-
Different to other methods used to assessing soil aggregate cally induced soil aggregate stability to the angle of internal friction
stability (Kemper and Rosenau, 1986; Le Bissonnais, 1996; Le (˚’) in view of quantifying vegetation effects on slope stability (Böll
Bissonnais and Arrouyas, 1997; Le Bissonnais et al., 2007) we and Graf, 2001; Frei, 2009). Based on triaxial compression tests
did not focus on small aggregates <2 mm and distinguish macro- using the same moraine, plant material, and sample sizes as in
(>250 ␮m) from microaggregates (<250 ␮m) as proposed by Tisdall the present study (Table 6), it was found that planted specimen
and Oades (1982). Instead, the starting soil material was com- (without mycorrhizal treatment) increased the angle of internal
posed of a loose mixture of grain sizes ≤10 mm and, in terms of friction by ∼5◦ (Graf et al., 2009). This gain in stability was compa-
soil aggregate stability, only components >20 mm were considered, rable to the increase in ˚ due to the compaction effect of pure
representing the next higher class of grain size from a geotechnical soil samples from a dry unit weight  = 15.7 to  = 18.9 kN m−3
point of view (Fig. 1). (Table 6). A similar effect was found in the present study for
This approach was chosen on the basis of soil mechanical consid- aggregate stability. The increase from 0.03 for pure soil with a
erations, particularly in order to be compatible with conventional dry unit weight  = 15.2 kN m−3 to 0.48 for compacted soil with
methods addressing soil stability, e.g. triaxial compression test, as  = 19.2 kN m−3 is approximately in the same order as the one
well as due to biological reasons. Superficial soil failure related for soil planted with mycorrhized alder with a dry unit weight
to heavy rainstorms is mainly due to water saturation and corre-  = 15.5 kN m−3 yielding an aggregate stability of 0.38 (Table 6;
sponding excessive pore water pressure (Iversen, 2000; Kuriakose Figs. 2 and 3). It seems evident from the data and physically plau-
et al., 2008) and, therefore, directly linked to the stability of the pore sible that the angle of internal friction (˚ ) as well as the aggregate
structure and the relevant soil matrix. In order to test the biological stability of pure moraine are each positively correlated with the
effects on aggregate stability in terms of resistance against slaking dry unit weight and, consequently, are for their part, too. This
(Le Bissonnais, 1996) a certain volume of the specimen is required coherence and the fact that ˚ of compacted moraine and planted
to fit for appropriate root development and to ensure the vadose moraine of lower dry unit weight correspond quite well, suggest
zone is representative in view of natural superficial soil conditions, the assumption that the stability effect of roots may be expressed
i.e. distribution of macro-, meso-, and micro-pores according to the as a virtual increase in . The same supposition may be made for soil
 F. Graf, M. Frei / Ecological Engineering 57 (2013) 314–323 321

aggregate stability of compacted moraine and moraine with myc- 5. Conclusions

orrhized plants. No such conclusion can be drawn for the planted
moraine without mycorrhiza for which aggregate stability was With respect to landscape restoration the positive influence of
significantly lower compared to the compacted and mycorrhized mycorrhizal fungi in different ecosystems has been found impor-
treatments (Table 5). Furthermore, a pairwise Wilcox Test of the tant in order to promote the relevant plant communities. However,
root data from both investigations (Graf et al., 2009; this study) on severely degraded soil, the natural recovery potential of the
revealed that root length per soil volume of the planted moraine symbiotic fungi is considerably impeded and the species com-
of Graf et al. (2009) significantly differed from the planted moraine position of adjacent intact regions may not necessarily fit the
of this study (p-value = 0.02) but not compared to the planted and requirements of the site to be re-colonised. Therefore, the applica-
mycorrhized samples (p-value = 0.62). Based on these relationships tion of indigenous plant species with carefully selected mycorrhizal
it seems, therefore, acceptable to compare the findings on the angle fungi is an excellent eco-engineering measure to initiate and accel-
of internal friction of planted moraine (Graf et al., 2009) with those erate the re-colonisation of slopes affected by superficial soil
of the aggregate stability of the samples with mycorrhized plants. failure.
By doing so, the joint stability effect of mycorrhiza and plants corre- Based on such an integral approach it has been demonstrated
sponds to a virtual increase in dry unit weight by ∼4 kN m−3 which with the present study that soil aggregate stability considerably
corresponds to an effective increase in the angle of internal friction increases and, from a soil mechanical perspective, likewise the soil
by ∼5◦ (Table 6). stability expressed by the angle of internal friction ˚ . The result-
That it is not only idle speculation is indicated by the technique ing higher resistance of the soil with respect to superficial failure
of estimating the angle of internal friction (˚ ) based on the grain is first and foremost due to better plant root performance and the
size distribution curve with corrections after Brinch Hansen (in direct fungal impact on micro- and macro-aggregation. The pos-
Lang and Huder, 1994). Given the distribution of the soil mate- itive correlation between ˚ and soil aggregate stability deduced
rial classified as GC-CL the estimation of ˚ for the loose material from their correlations with the dry unit weight  is suggesting
is ∼34◦ . With the proposed surplus of up to 6◦ for compaction we to estimate and calculate biological effects related to slope sta-
finally have ˚ ≈ 40◦ which is in line with the results of both the tri- bility based on soil aggregation in place of the demanding and
axial compression tests and, linked by the dry unit weight , with time-consuming determination of the angle of internal friction ˚ .
the soil aggregate stability analysis (Table 6). However, what was demonstrated for one soil type (GC-CL) and the
It is subject to speculation if the angle of internal friction (˚’) natural “restoration package” Alnus icana with Melanogaster var-
will further increase in samples with mycorrhized plants. There is, iegatus s.l. needs to be verified with other soils and plant-fungus
however, some evidence to suggest it. Michalowski and Čermák combinations.
(2002, 2003) found that the reinforcing effect of fibres in sand is If appropriate, the application of carefully selected mycorrhizal
positively correlated with the length of the fibres. Furthermore, inoculi in eco-engineering will be doubtlessly an important step
Zhang et al. (2006) demonstrated a positive correlation between towards more sustainability as well as easier integration into engi-
the shear strength of sand and the complexity of 3D structures neering concepts and risk management. Moreover, the biological
added. Graf et al. (2009) reported the highest mobilised shear contribution to slope stability will finally get its true worth in slope
strength ˚’ = 41.03◦ (1.64◦ above the mean value, Table 6) of stabilisation and protection against superficial soil failure.
planted moraine from the sample with the highest root length
(2.17 cm cm−3 ) which was about twice the mean value (Table 6).
Acknowledgements
They also observed higher ramification degrees of lateral roots in
samples with higher root length per soil volume. Therefore, mycor-
We thank Albert Böll for his helpful suggestions and comments
rhiza may impact on ˚ at least indirectly by its effect on root
related to soil mechanics. Furthermore, we appreciate the sugges-
growth, positively impinge on both root length and ramification.
tions of two anonymous reviewers. The PhD project of co-author
In the course of this argumentation, the poor aggregate sta-
Martin Frei, who provided the data, was funded by the Swiss Federal
bility of the planted samples compared to them with mycorrhiza
Office for the Environment FOEN.
can be mainly explained by the 2.5 times lower root length. How-
ever, under consideration of the method applied for quantifying soil
aggregate stability, the direct fungal effect should not be ignored. It Appendix A. Supplementary data
is not just that root length was less than half and ramification low
without mycorrhiza. It is also the lack of a fine-meshed hyphal net- Supplementary data associated with this article can be
work firmly adhering soil among as well as in the space in between found, in the online version, at http://dx.doi.org/10.1016/
roots and, therefore, eliminating spatial constraints with regard j.ecoleng.2013.04.037.
to a continuous three-dimensional structure of stable micro- and
macro-aggregates (Bogeat-Triboulot et al., 2004). Due to the ten-
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