Chemical and biological characterization of dissolved organic
matter from silver fir and beech forest soils
D. Pizzeghello
a
, A. Zanella
b
, P. Carletti
c
, S. Nardi
a,*
a
Dipartimento di Biotecnologie Agrarie, Universita` di Padova, Agripolis, Viale dell’Universita` 16, 35020 Legnaro, Padova, Italy
b
Dipartimento Territorio e Sistemi Agro-Forestali, Universita` di Padova, Agripolis, Viale dell’Universita` 16, 35020 Legnaro, Padova, Italy
c
Centro di Ecologia Alpina, 38040 Viote del Monte Bondone, Trento, Italy
Received 17 October 2005; received in revised form 28 February 2006; accepted 1 March 2006
Available online 18 April 2006
Abstract
Despite a growing attention to the dissolved organic matter (DOM) in terrestrial ecosystems and evidence of the fact that vegetation
affects the quality of both undissolved and dissolved organic matter in soil, the role of DOM as a biological indicator is still poorly under-
stood. In this work, the fertility of 59 sites, divided into eight key alliances of the order Fagetalia sylvaticae Pawl., was studied considering
chemical and biological parameters such as soil DOM, hormone-like activity, low-molecular-weight (LMW) aliphatic and phenolic acids,
and floristic data.
Both non-parametric tests and principal component analysis (PCA) revealed differences between silver fir and beech forests and within
each type of forest. There were also differences between neutrophilous and acidophilous types. What’s more, PCA reveals the dominance
of the auxin (IAA)-like activity, and of some phenolic acids in distinguishing the acidophilous beeches (ACI) form the other types,
whereas the gibberellin (GA)-like activity is more relevant in neutrophilous conditions such as thermophilous (THE) and mesophilous
(MESO) beeches and montane (MO), high montane (HMA), high montane (HMC) silver fir forests. The GA-like activity is also related
to the succinic, fumaric, malonic, and
L
-malic acids in the MO, HMA and HMC silver fir forests. Moreover, the role of LMW aliphatic
acids in mobilizing the hormone-like activity, which improves forest growth, is stressed.
The growth of seedlings of Picea abies was influenced by the phenolic acid content. At concentrations between 1 and 100 lM, phen-
ylacetic and protocatechuic acids inhibited root growth to the same extent as indoleacetic acid, while p-hydroxybenzoic acid had a stim-
ulating effect comparable to that of gibberellic acid.
The aliphatic and phenolic acids appear to be related to plant strategies that influence soil fertility affecting plant growth through
rhizodeposition. The role of LMW aliphatic and phenolic acids as molecular markers of ecosystem function is noted.
2006 Elsevier Ltd. All rights reserved.
Keywords: Silver fir; Beech; Low-molecular-weight aliphatic and phenolic acids; Hormone-like activity; Bioassay on Norway spruce seedlings
1. Introduction
Dissolved organic matter (DOM) is probably the most bio-
available fraction of soil organic matter (
), which contributes significantly to nutrient
cycles (
) and is important in the following
contexts:
1. it is a major mode of nitrogen and phosphorus export in
many ecosystems;
0045-6535/$ - see front matter
2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2006.03.001
Abbreviations: DOM, dissolved organic matter; LMW, low-molecular-
weight; MO, montane; HMA, high montane with Adenostylo; HMC, high
montane caricetosum; AC, acidophilous with Pyrola; A, acidophilous with
Carici; THE, thermophilous; MESO, mesophilous; ACI, acidophilous
beech forests; IAA, auxin-like activity; GA, gibberellin-like activity; oxa,
oxalic acid; tar, tartaric acid;
L
-mal,
L
-malic acid; mal, malonic acid; suc,
succinic acid; fum, fumaric acid; total AA, total aliphatic acids; proto,
protocatechuic acid; proto al, protocatechuic aldehyde; p-hydro, p-hydr-
oxybenzoic acid; van, vanillic acid; val, vanilline; phen, phenylacetic acid;
ben, benzoic acid; total PA, total phenolic acids.
*
Corresponding author. Tel.: +39 049 8272911; fax: +39 049 8272929.
E-mail address:
(S. Nardi).
www.elsevier.com/locate/chemosphere
Chemosphere 65 (2006) 190–200
2. it plays an important role in determining the balance
and accumulation of nitrogen, phosphorus, and possibly
of carbon, in long-term soil development;
3. being DOM normally acidic, and displaying a powerful
metal complexing activity, it is deeply involved in min-
eral weathering, metal toxicity mediation, and metal
export, i.e., to water bodies;
4. it provides a potential source of carbon for microbial
growth.
Many authors suggest that tree species and ground veg-
etation affect the quality and quantity of both undissolved
and dissolved organic matter in the soil (
1998; Hongve et al., 2000; Kaiser et al., 2001; Park et al.,
2002
), while others support the view that tree species have
little effect on DOM composition and chemical reactivity
(
). Vegetation nonetheless remains
an important source of soil DOM as its growth, death,
and decomposition release organic substances. In fact,
from 22% to 41% of the C in the freshly fallen autumn leaf
litter of a deciduous forest is water soluble (
).
Moreover, secondary metabolites found in trees, espe-
cially phenolic compounds, can have fundamental effects
on C and N dynamics in forest soils (
).
Phenolics are thought to arise from decomposing plant
matter, some can be synthesized by soil microorganisms
and some released as plant root exudates. Plants have
repeatedly recruited phenolics as signal molecules to either
facilitate or discourage interactions with other organisms
(
). In soil, low-molecular-weight (LMW) phe-
nolic acids may reach concentrations sufficient to directly
or indirectly influence plant growth. In fact, at concentra-
tions between 0.1 and 1 mM, many phenolic compounds
are toxic to plants, and to seedlings in particular, while
concentrations ranging from 0.1 lM to 0.1 mM have gen-
erally less apparent toxic effects and there is evidence that
the growth of some plant species may even be enhanced
under certain conditions.
Notwithstanding the increasing attention to the role of
DOM in terrestrial ecosystems (
et al., 2000, 2003; Kalbitz and Kaiser, 2003; Qualls et al.,
2002; van Hees et al., 2005
), its effect on plant growth is still
poorly understood.
The aim of the present project was to study the chemical
parameters of DOM from forest soils and to evaluate the
differences in the biochemical activity of DOM in relation
to the different forest associations. For such purpose, 59
sites, including 32 silver fir and 27 beech forest sites, were
considered. Plant cover, as well as the height and diameter
of the dominant trees were measured. DOM values of the
59 soils were characterized by LMW organic aliphatic and
phenolic acids detection, using an HPLC diode-array tech-
nique. The biological properties of the DOM were evaluated
by determining the auxin (IAA) and gibberellin (GA)-like
activities, since indoleacetic acid and gibberellic acid are
important in controlling seed germination as well as plants’
adaptation to environmental stress. In addition, the effect of
phenolic acids on root and shoot growth of Norway spruce
(Picea abies L. Karst.) seedlings was also determined.
2. Materials and methods
2.1. Study area
The silver fir and beech forest study area was located in
the Autonomous Province of Trento (north-east Italy) and
it was widely described in previous works (
). Briefly, from the statistical processing
of the floristic data, eight large groups corresponding to
key alliances of the order Fagetalia sylvaticae Pawl. were
considered (
), i.e.,
• the alliance Fagion sylvaticae with the association
Cardamino pentaphylli–Abietetum albae (
(montane silver fir forests, MO),
• the alliance Fagion sylvaticae with the association Ade-
nostylo glabrae–A. albae (
) (high montane
silver fir forests, HMA),
• the alliance Fagion sylvaticae with the association
A. glabrae–A. albae and the sub-association A. glabrae–
Table 1
Soil classification and parameters of dominant trees of the 59 forest sites
n
Cover
Type
h (m)
B
(m)
Age (y)
Soil classification
8
Silver fir
MO
27.6
0.45
103
Calcaric PHAEOZEM
4
Silver fir
HMA
31.7
0.50
101
Haplic PHAEOZEM
8
Silver fir
HMC
26.7
0.42
136
Chromic LUVISOL
6
Silver fir
AC
24.5
0.36
117
Dystric CAMBISOL
6
Silver fir
A
23.9
0.32
116
Haplic PODZOL
8
Beech
THE
15.9
0.23
59
Skeletic–Calcaric CAMBISOL, Eutric CAMBISOL,
Calcaric CAMBISOL
14
Beech
MESO
17.2
0.26
62
Eutric CAMBISOL, Skeletic–Calcaric CAMBISOL,
Chromic LUVISOL, Haplic LUVISOL, Luvic PHAEOZEM,
Skeletic–Calcaric PHAEOZEM
5
Beech
ACI
18.2
0.27
72
Dystric CAMBISOL, Haplic PODZOL
Silver fir types were montane (MO), high montane with Adenostylo (HMA), high montane with Abietetum caricetosum (HMC), acidophilous with
Calamagrostis (AC), acidophilous (A), while beech types were thermophilous (THE), mesophilous (MESO) and acidophilous (ACI).
n, number of sites; h, height; B, diameter.
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
191
Abietetum caricetosum albae (
) (high mon-
tane silver fir forests, HMC),
• the alliance Luzulo–Fagion with the association Luzulo
niveae–Abietetum (
) (acidophilous silver fir
forests, AC),
• the alliance Luzulo–Fagion with the association Pyrolo–
A. albae (
) (acidophilous silver fir forests, A),
• the alliance Cephalanthero–Fagion with the association
Carici albae–Fagetum. (thermophilous beech forests,
THE),
• the alliance Fagion sylvaticae with the associations Den-
tario pentaphylli–Fagetum and Galio-odorati–Fagetum
(mesophilous beech forests, MESO),
• the alliance Luzulo–Fagion with the association L.
niveae–Fagetum (acidophilous beech forests, ACI) (
The 59 sites were chosen within these forested areas and
the height, diameter and age of the dominant trees were
measured (
). At the same sites, 59 typical soil sam-
ples were obtained by excavating a soil pit c. 1 m
2
in sur-
face area down to a depth that never exceeded 1.5 m.
Triplicate samples were taken from each A horizon. After
screening for large rock fragments, the soil samples were
placed in plastic bags, kept at a temperature of 4
C and
taken to the laboratory for DOM extraction and character-
ization. The soils were classified according to the
system criteria (
2.2. Dissolved organic matter extraction
DOM was extracted from the fresh A horizon soil sam-
ples using double-deionized water with a solid/volume ratio
of 1:2 (
). The suspensions were shaken for
2 h at room temperature in a N
2
atmosphere, and then cen-
trifuged at 10
C and 7000g for 15 min. The extracts were
filtered on microfiber glass filters (Whatman, Maidstone,
England), then on nylon 0.45 lm filters (Millipore, Milford,
MA, USA). The dissolved organic carbon (DOC) content
was assayed by dichromate oxidation (
).
2.3. Low-molecular-weight organic acid determination
To determine the LMW organic acids, an aliquot of
DOM was preliminarily purified in an anion exchange
solid-phase extraction column (SAX, Alltech, Deerfield,
IL). Anionic fractions were eluted with 500 mM H
2
SO
4
.
Free organic acids were separated by HPLC using a 400
Perkin–Elmer high-pressure pump (Perkin–Elmer, Nor-
walk, Connecticut) equipped with a 234 Gilson auto-injec-
tor (Gilson, Middlestone, WI) and an LC 90 Perkin–Elmer
variable-wavelength UV detector set at 210 nm, with 10-ll
flow cell. Ionic-exchange chromatography was carried out
using an HPX-87-H Aminex column (Biorad, Richmond,
CA) (300 mm
· 7.8 mm i.d.) and isocratic elution with
6 mM H
2
SO
4
as mobile phase (0.4 ml min
1
) at room
temperature (
). The reference standards
were supplied by Sigma (Sigma, St. Louis, MO).
2.4. Low-molecular-weight phenolic acids
To measure the phenolic acids, a standard volume of
DOM was acidified (pH = 3.0) with acetic acid and then
extracted three times with ethyl acetate. After evaporating
to dryness, the residues were redissolved in water. These
water solutions were used for phenolic HPLC determi-
nation and quantification. Protocatechuic acid, proto-
catechuic aldehyde, p-hydroxybenzoic acid, vanillic acid,
vanilline, phenylacetic acid and benzoic acid were sepa-
rated by HPLC and recorded by a diode-array detector
(Hewlett Packard, Palo Alto, CA), with a 100-ll flow cell.
Reversed-phase chromatography was done in a Luna 5 lm
C-18 end-capped analytical column (250 mm
· 4.6 mm,
i.d., Phenomenex, Torrance, CA) and isocratic elution with
water–acetic acid–n-butanol (Fluka, Buchs, Switzerland)
(342:1:11, v/v/v) and a flow rate of 1.2 ml min
1
). The reference standards were supplied
by Sigma. Semiquantitative results were obtained because
only the identified phenolic acids were integrated.
2.5. Hormone-like activities
The auxin and gibberellin-like activities of the DOM
were assessed by checking the growth reduction of water-
cress (Lepidium sativum L.) roots and the increase in the
length of lettuce (Lactuca sativa L.) shoots, respectively
(
). Watercress and lettuce seeds were surface-
sterilized by immersion in 8% hydrogen peroxide for
15 min. After rinsing five times with sterile distilled water,
10 seeds were placed on sterile filter paper in a sterile Petri
dish. For watercress, the filter paper was wetted with 1.2 ml
of 1 mM CaSO
4
(control); or 1.2 ml of 20, 10, 1 and
0.1 mg l
1
indoleacetic acid (Sigma) to obtain the calibra-
tion curve; or 1.2 ml of 10, 5, 2.5, 1, 0.5, 0.2 and
0.1 mg C l
1
of the extracted DOM. For lettuce, the exper-
imental design was the same as for watercress except that
the sterile filter paper was wetted with 1.4 (not 1.2) ml of
1 mM CaSO
4
(control) or of 10, 5, 2.5, 1, 0.5, 0.2 and
0.1 mg C l
1
of the extracted DOM, and the calibration
curve was a progression of 100, 10, 1 and 0.1 mg l
1
gibber-
ellic acid (Sigma). The seeds were germinated in the dark at
25
C in a germination room. After 48 h for watercress and
72 h for lettuce, the seedlings were removed and the root or
shoot lengths were measured (
). The results
were recorded as concentrations of indoleacetic acid or
gibberellic acid of activity equivalent to 1 mg C l
1
of
DOM.
2.6. Bioassay on Norway spruce seedlings
According to
, laboratory bio-
assays are important tools for understanding a particular
component of allelopathy, but assays on artificial sensitive
192
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
species such as lettuce and watercress must be followed up
with those on natural species in the ecosystem studied. We
consequently tested the effect of the phenolic acids, at a
range of concentrations similar to those found in the soil
solution, on the root and shoot growth of Norway spruce
seedlings, as this species was present in all of our forest
types. To obtain a functional interpretation, the effect of
the phenolic acids was compared with that of some plant
hormones.
Norway spruce (P. abies L. Karst.) seeds were surface-
sterilized by 30 min contact in 8% hydrogen peroxide
solution. After rinsing 5–6 times with sterile distilled water,
seeds were placed in Petri dishes containing Whatman No.
3 filter papers wetted with 1.4 ml of protocatechuic acid, or
protocatechuic aldehyde (0.1–50 lM), or p-hydroxybenzoic
acid (1–350 lM), or vanillic acid (0.1–100 lM), or vanilline
(0.1–100 lM), or phenylacetic acid, or benzoic acid (1–
100 lM) as phenolic acids. We considered a progression
of 20–0.1 mg l
1
indoleacetic acid (Sigma) for the auxin-
like calibration curve, and 100–0.1 mg l
1
gibberellic acid
(Sigma) for the gibberellin-like calibration curve, and also
a 1 mM CaSO
4
solution for control purposes. The seeds
were germinated in the dark at 25
C in a germination
room. Seedling root or shoot lengths were measured at
12 d old. The values obtained were the means of 20 samples
and three replications, with the standard errors always
6
5% of the mean.
2.7. Statistical analyses
Goodness-of-fit to normal distribution was tested for all
variables using the Shapiro–Wilks W-test. None of the
data-set was normally distributed (P < 0.01). Non-para-
metric Kruskal–Wallis H-test and its multiple comparison
extension were therefore used to test for significant differ-
ences between forest types (
). Correlations
between variables were determined using Spearman’s coef-
ficient. To clarify the structure of these interdependences,
we performed a joint principal components analysis
(PCA) of our data on 14 variables. PCA is a multivariate
technique to investigate relationships among several quan-
titative variables. Given a data-set with p numeric vari-
ables, p PCs can be computed. Each PC is a linear
combination of the original variables, with the coefficients
equal to the eigenvectors (loading coefficients) of correla-
tion matrix. The eigenvectors are usually taken with unit
length and they are orthogonal, so PCs are jointly uncorre-
lated. The PCs are sorted by decreasing order of the eigen-
values, which are equal to the variances of the components.
Therefore, the purpose of PC analysis (
) is to
derive a small number of linear combinations (Principal
Components) of a set of variables that retain as much of
the information in the original variables as possible. PCs
are often followed by rotation of the components to aid
their interpretation. Fourteen standardized variables were
Table 2
Chemical and biological characteristics of soils from the silver fir and beech forest studied
Silver fir
Beech
MO
HMA
HMC
AC
A
THE
MESO
ACI
1
2
3
4
5
6
7
8
pH
6.46a
6.37a
4.50b
3.83b
6.67a
6.47a
4.07b
DOC (%)
0.160a
0.066b
0.136a
0.052b
0.104a
0.048b
0.058b
0.070b
oxa (lM)
829d
615e
872d
1484c
5356a
2121b
166f
506e
tar (lM)
1113b
1162b
1060b
1384b
5820a
472d
376d
714c
L
-mal (lM)
847c
262d
1558b
934c
2149a
166d
87e
51e
mal (lM)
343a
137c
336a
220b
394a
133c
112c
87c
suc (lM)
1272b
1231b
1656a
603c
424d
205e
170e
175e
fum (lM)
33a
14b
22b
13b
18b
12b
7c
3c
AA (lM)
4436b
3420c
5503b
4636b
14160a
1199d
919d
1536d
proto (lM)
3.43c
4.75c
9.82c
24.78b
36.80b
2.02c
6.68c
60.46a
proto al (lM)
0.62c
0.70c
1.54b
3.03a
0.76c
0.62c
0.81c
3.01a
p-hydro (lM)
36.60c
31.67c
45.07c
17.70d
2.08e
78.85b
96.57a
15.30d
van (lM)
20.10b
25.57b
44.65a
28.70
4.20d
10.95c
21.70b
15.74bc
val (lM)
3.36a
3.15a
4.15a
3.90a
1.62b
0.97c
3.33a
2.80ab
phen (lM)
9.98c
9.80c
10.97c
23.25b
29.48b
4.25d
14.66c
58.68a
ben (lM)
4.94c
4.42c
6.18c
12.42b
14.62b
5.92c
6.94c
31.82a
PA (lM)
79d
80.25d
122c
114c
89.60d
103.75c
151b
187.80a
IAA
0.024d
0.015d
0.012d
0.044c
0.097b
0.015d
0.041c
0.317a
GA
0.008d
0.010d
0.069b
0.029c
0.008d
0.097a
0.050b
0.017c
Silver fir types were montane (MO), high montane with Adenostylo (HMA), high montane with Abietetum caricetosum (HMC), acidophilous with
Calamagrostis (AC), acidophilous (A), while beech types were thermophilous (THE), mesophilous (MESO) and acidophilous (ACI).
DOC, dissolved organic C; oxa, oxalic acid; tar, tartaric acid;
L
-mal,
L
-malic acid; mal, malonic acid; suc, succinic acid; fum, fumaric acid; AA, total
aliphatic acids; proto, protocatechuic acid; proto al, protocatechuic aldehyde; p-hydro, p-hydroxybenzoic acid; van, vanillic acid; val, vanilline; phen,
phenylacetic acid; ben, benzoic acid; PA, total phenolic acids; IAA, auxin-like activity of DOM expressed as concentration of indoleacetic acid activity
equivalent to 1 mg C l
1
DOM; GA, gibberellin-like activity of DOM expressed as concentration of gibberellic acid of activity equivalent to 1 mg C l
1
DOM.
*
Values in the same row, followed by the same letter, are not statistically different at P 6 0.05.
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
193
Table 3
Significance (Kruskal–Wallis test H) of the variables distinguishing the 59
sites by soil pH, main vegetation (silver fir and beech) and floristic data
(eight types: MO, HMA, HMC, AC, A, THE, MESO and ACI)
Variables
Soil pH
Silver to beech
Eight types
pH
0.000
0.508
0.000
DOC
0.451
0.188
0.257
oxa
0.003
0.000
0.000
tar
0.008
0.000
0.000
L
-mal
0.056
0.001
0.001
mal
0.905
0.000
0.008
suc
0.124
0.000
0.000
fum
0.275
0.000
0.001
AA
0.043
0.000
0.000
proto
0.000
0.376
0.000
proto al
0.001
0.903
0.024
p-hydro
0.000
0.016
0.000
van
0.020
0.473
0.011
val
0.743
0.292
0.061
phen
0.000
0.490
0.000
ben
0.000
0.461
0.000
PA
0.226
0.055
0.097
IAA
0.000
0.772
0.000
GA
0.139
0.0046
0.000
*
P indicates significant differences when 60.05.
Table 4
Loadings of chemical and biochemical properties on the axes identified by
principal components analysis of their values in soils under the different
silver fir and beech forests studied
Variables
PC 1
PC 2
PC 3
PC 4
pH
0.59
0.23
0.10
0.66
oxa
0.14
0.19
0.15
0.90
tar
0.13
0.21
0.11
0.89
L
-mal
0.08
0.55
0.11
0.56
mal
0.03
0.72
0.18
0.34
suc
0.28
0.69
0.05
0.03
fum
0.21
0.79
0.01
0.13
proto
0.86
0.17
0.04
0.34
p-hydro
0.11
0.12
0.56
0.44
van
0.01
0.18
0.78
0.23
phen
0.93
0.01
0.07
0.06
ben
0.91
0.06
0.12
0.09
GA
0.33
0.35
0.66
0.05
IAA
0.88
0.15
0.09
0.02
Explained variance (%)
32.9
23.3
9.9
8.4
Cumulated explained
variance (%)
32.9
56.2
66.1
74.5
-2
0
2
PC 1 32.9 %
-2
0
2
PC 2 23.3%
1
1
1
1
1 1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
6
6
6
6
7
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
pH
oxa
tar
l-mal
mal
suc
fum
proto
p-hydro
van
phen
ben
ga
iaa
Fig. 1. Score plot of PCA showing the separation of the 59 silver fir and beech forest stands along principal components (PC) 1 and 2 and loading values
for the fourteen variables. PCA was performed using rotation of the first two components. The different forest types were: (1) montane silver fir, (2) high
montane with Adenostylo silver fir, (3) high montane with Abietetum caricetosum silver fir, (4) acidophilous with Calamagrostis silver fir, (5) acidophilous
silver fir (A), thermophilous beech (6), mesophilous beech (7) and acidophilous beech (8).
194
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
submitted to the PC analysis, using the factor procedure of
the software package STATISTICA. Rotated orthogonal
components (varimax method of rotation) were extracted
and the relative scores were determined. The number of
considered components was determined so that the amount
of variance explained by the eigenvalues included a suffi-
cient part of total variability.
3. Results
The mean values of the chemical and biochemical
parameters for each type are shown in
. As regards
DOC quantity, the silver fir types are endowed with a
higher content with respect to beech, and no trend is evi-
denced from the neutrophilous MO, HMA and HMC to
the acidophilous AC and A. On the contrary, the DOC
content in the beech types rises from the thermophilous
THE to the acidophilous ACI. Considering the LMW
organic acids, total aliphatic acids (AA) are higher in the
silver fir than in the beech types. In the silver fir forests
both oxalic and
L
-malic acids are higher in AC and A than
MO, HMA and HMC, while in the beech forests the
amount of these acids decreases from THE to ACI. Among
the aliphatic acids, oxalic, tartaric,
L
-malic and succinic
acids are the dominant LMW organic acids in the studied
soils. Tartaric acid is always higher in acidic AC, A and
ACI conditions than basic MO, HMA, HMC, THE and
MESO conditions, while the succinic acid is higher in basic
than acidic conditions. The total phenolic acid content
(PA) is lower in silver fir types than in beech types. In these
forests, protocatechuic, benzoic and phenylacetic acids are
higher in acidic than in basic conditions, while p-hydroxy-
benzoic acid proves higher in neutral than in acidic
conditions.
The auxin-like activity (IAA) of DOM (
) both in
silver fir and beech types is high in acid conditions, while
the gibberellin-like (GA) activity is generally high in neu-
trophilous conditions.
Using the Kruskal–Wallis H-test to examine overall dif-
ferences between the eight types of forest (
, 3rd col-
umn), pH, LMW organic acids (oxalic, tartaric,
L
-malic,
malonic, succinic, fumaric), phenolic acids (protocatechuic,
0.2
0.4
0.6
0.8
0.1
1
10
100
Indoleacetic acid (ppm)
Ipocotyl/ipocotyl control
0.5
1
1.5
2
2.5
1
10
100
Phenylacetic acid (µM)
Ipocotyl/ipocotyl control
Fig. 2. Effect of indoleacetic and phenylacetic acids on root length of 12-d-old Norway spruce seedlings. Vertical bars indicate one SE to either side of the
mean.
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
195
protocatechuic aldehyde, p-hydroxybenzoic, vanillic, phen-
ylacetic and benzoic), IAA and GA all vary significantly.
Grouping the eight types according to the main vegetation
(
, silver to beech), all the aliphatic acids, the
p-hydroxybenzoic acid and GA are significant in distin-
guishing the beech woods from the silver fir woods. Upon
grouping by soil pH (
), neutrophilous and acidoph-
ilous types differ in relation to pH, oxalic, tartaric, AA,
phenolic acids and IAA.
The matrix of correlation (data not shown) indicates that
the IAA-like activity of DOM correlates positively with
oxalic (0.880, P 6 0.05), tartaric (0.885, P 6 0.05) and AA
(0.764, P 6 0.05), and with some phenolics, such as proto-
catechuic acid (0.562, P < 0.05), phenylacetic acid (0.626,
P 6 0.05) and benzoic acid (0.603, P 6 0.05). GA-like activ-
ity correlated positively with p-hydroxybenzoic acid (0.311,
P 6 0.05). The diameter of the dominant trees correlated
directly with succinic (0.713, P 6 0.05), fumaric (0.630,
P 6 0.05), tartaric (0.508, P 6 0.05) and oxalic (0.501,
P 6 0.05) acids and inversely with the IAA-like activity
( 0.469, P 6 0.05). The height of the dominant trees corre-
lates positively with succinic (0.688, P 6 0.05), fumaric
(0.611, P 6 0.05) and tartaric (0.562, P 6 0.05) acids.
From the PCA four main factors identified together
account for 74% of the variance (
also
listed the loading values of the soil DOM characteristics
considered for each factor. PC axis 1, which accounts
for 32.9% of the total variance, exhibits close negative
correlation with phenylacetic acid, benzoic acid, IAA
and protocatechuic acid, and a somewhat lower positive
correlation with the pH of DOM. PC axis 2 accounts
for 23% of the total variance and it is defined mainly
by fumaric, malonic, succinic, and
L
-malic acids. PC 3
and 4 account for only 9.8% and 8% of the total vari-
ance, respectively. The main defining properties are vanil-
lic acid and GA for PC 3, and oxalic and tartaric acids
for PC 4.
Plotting PC 1 and PC 2 (
) it is evident that the
ACI (8) beeches are strictly separated from all the other
forest types and that the acidophilous ACI (8), AC (4)
and A (5) are located on the left side of the graph, while
neutrophilous MO (1), HMA (2), HMC (3), THE (6)
and MESO (7) types are on the right side (
). More-
over, the silver fir types (1, 2, 3, 4, 5) prevail in the mid-
dle-top of the graph while the beech (6, 7, 8) types are in
the middle-bottom.
0.65
0.7
0.75
0.8
0.85
0.9
0.95
0.1
1
10
100
Protocatechuic acid (µM)
Ipocotyl/ipocotyl control
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.1
1
10
100
Vanilline (µM)
Ipocotyl/ipocotyl control
Fig. 3. Effect of protocatechuic acid and vanilline on root length of 12-d-old Norway spruce seedlings. Vertical bars indicate one SE to either side of the
mean.
196
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
Both non-parametric tests and PCA revealed differences
between silver fir and beech forests, as well as when the
neutrophilous types were compared with the acidophilous.
In particular, PCA reveals the influence of the IAA-like
activity, and of some phenolic acids in distinguishing the
acidophilous beeches form the other types. The GA-like
activity is more relevant in neutrophilous conditions, espe-
cially in the THE (6) and MESO (7) beeches, while succi-
nic, fumaric, malonic, and
L
-malic acids are important in
distinguishing the MO (1), HMA (2) and HMC (3) from
the other types.
The phenolic acids influenced the growth of seedlings of
Norway spruce. In detail, phenylacetic acid and proto-
catechuic acid, at concentrations between 1 and 100 lM,
and vanilline, at a concentration between 1 and 10 lM,
inhibited root growth to an extent comparable to that of
indoleacetic acid (
). As for shoot growth, vanil-
lic acid had a stimulant effect at a concentration between 0.1
and 50 lM (
), as did vanilline at a concentration
between 1 and 100 lM and p-hydroxybenzoic acid at a con-
centration between 1 and 100 lM, yielding a response com-
parable to that of gibberellic acid (
).
4. Discussion
There is a lack of knowledge concerning the effect of dif-
ferent tree species on the amount and quality of DOC.
found the highest amounts lea-
ched from deciduous litter, whereas
extracted higher DOC amounts from spruce forest floor
than from beech, oak and grand fir.
did not find a general trend when compar-
ing DOC concentrations of coniferous forests with those of
deciduous temperate forests.
In our study, the higher DOC content present in silver
fir with respect to beech is not surprising, as beech is a
deciduous species. It has been shown that the average age
of C residues in freshly fallen deciduous litter and in the
soil is lower than that observed with conifers such as spruce
and silver fir (
1
1.1
1.2
1.3
1.4
0.1
1
10
100
Gibberellic acid (ppm)
Epicotyl/epicotyl control
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
0.1
1
10
100
Vanillic acid (µM)
Epicotyl/epicotyl control
Fig. 4. Effect of gibberellic acid and vanillic acid on shoot length of 12-d-old Norway spruce seedlings. Vertical bars indicate one SE to either side of the
mean.
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
197
In addition we have found that the DOM of silver fir
forests is endowed with a higher LMW aliphatic acid con-
tent and less phenolic acids, GA and IAA-like activities
than that of beech forests.
In accordance with our results,
showed that the concentration of LMW organic acids
was higher in the soil solution under conifers as compared
to under deciduous species. He also showed a pattern of
LMW organic acids similar to that found in our soils, con-
sisting of oxalic,
L
-malic, malonic and succinic acids. The
quantitative differences between our study and that of Stro-
bel are related to the different horizons considered: Strobel
considered the O horizon, whereas we considered the A
horizon.
It is puzzle to justify the correlation between aliphatic
acids and the height and diameter of trees as plant growth
is a metabolic process related to various physiological
events. It is known that LMW organic acids, i.e., oxalic,
tartaric and succinic acids, secreted by plant root exudates
break down HS from high to low molecular size. Thus, the
resulting LMW humic substances can get into the root sys-
tem and interfere with plant metabolism (
). In the shift from high to low molecular size, the
so-called depolycondensation process, organic acids release
hormone-like activity from humic matter, that is not bio-
available in the polymer, and that markedly affects plant
growth (
).
Phenolic acids in soils, depending on their chemical state,
concentrations, and the organisms involved, can have no
effect, a stimulatory effect, or some inhibitory effects on cer-
tain plants or microbial processes. Some phenolic acids
identified as potential allelophatic agents are: proto-
catechuic acid, hydroxybenzoic acid, vanillic acid and p-
coumaric acid (
). They influence membrane
perturbation which is followed by a cascade of physiologi-
cal effects that include improvement of plant–water rela-
tionships, stomatal function and rate of photosynthesis
and respiration. These phenolics also interact with several
phytormones and enzymes determining a different biosyn-
thesis and flow of carbon into metabolites (
).
In our soils the pattern of phenolic acids differed both
with the vegetation and with the soil pH as evidenced by
the Kruskal–Wallis test. Phenolic acids such as phenylace-
tic, benzoic and protocatechuic are dominant in acidic soils
0.8
0.9
1
1.1
1.2
1.3
0.1
1
10
100
Vanilline (µM)
Epicotyl/epicotyl control
1
1.1
1.2
1.3
1
10
100
1000
p-Hydroxybenzoic acid (µM)
Epicotyl/epicotyl control
Fig. 5. Effect of vanilline and p-hydroxybenzoic acid on shoot length of 12-d-old Norway spruce seedlings. Vertical bars indicate one SE to either side of
the mean.
198
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
while p-hydroxybenzoic is important in neutral soils. As
also revealed by PC analysis these phenolic acids are
strictly linked to the hormone-like activity: the phenylace-
tic, benzoic and protocatechuic acids are important deter-
minants of IAA-like activity, whereas p-hydroxybenzoic
acid is determinant for the GA-like activity.
The regeneration of natural forests is affected by LMW
phenolic acids of soil and soil solution (
). In our soils the LMW phenolic acids of
DOM affected the Norway spruce seedlings’ growth. In
fact, the roots were shorter than the control when seedlings
were incubated with indoleacetic acid, phenylacetic acid,
protocatechuic acid and vanilline, thereby demonstrating
IAA-like activity. The greatest shoot length was always
induced by gibberellic acid, p-hydroxybenzoic acid, vanillic
acid and vanilline, demonstrating a gibberellin-like activity.
Among the phenolic acids, phenylacetic acid has been
reported to be produced by Frankia strains and its presence
in the rhizosphere has been shown to affect the hormone
balance crucial to root tissue by changing the cytokinin/
auxin ratio of root cortical cells (
).
In a previous study (
) we deter-
mined a discriminant function which well distinguished
the 59 soils by the properties related to their organic matter,
humic matter and humic substance’s hormone-like activity.
In that model the hormone-like activity of HS produced one
of the most important variables. In our study the figure of
PCA showed the difference between silver fir and beech for-
ests. The acidophilous beeches (ACI) are strictly linked to
IAA-like activity and to phenylacetic, benzoic and proto-
catechuic acids. THE and MESO beeches are endowed with
a high GA-like activity. AC and A silver fir types are in a
intermediate position between IAA and GA-like activity,
while in the MO, HMA and HMC silver firs the GA-like
activity appear to be linked to aliphatic acids such as fuma-
ric, malonic,
L
-malic and succinic.
In conclusion, the results showed that the composition
of DOM varied with the vegetal association. The slow evo-
lution of organic matter in the silver fir forests determined
a DOM rich in C and aliphatic acid content, whilst the fast
organic matter turnover in the beech forests determined a
low content of organic C and aliphatic acids and a high
content of phenolic acids and of the hormone-like activity.
The link between DOM organic acids and plant growth
may be the result of the different plant strategies that,
through
rhizodeposition,
influence mineral nutrition.
DOM characteristics ultimately offer insights into the
mechanisms of the ecosystem and indicate that the growth
of different forests could be strongly linked to the presence
of soluble compounds.
Acknowledgements
We thank the Forestry Commission of the Autonomous
Province of Trento for helping us choose the study sites,
and M.S. Calabrese, G. Sartori, A. Mancabelli, M. Tomasi
and C. De Siena for field sampling and soil classification.
Special thanks go to F. Morari for statistical help in the
PC analysis and to Thomas Russel and A. Squartini for
improving the English of the manuscript. This work was
funded by the Autonomous Province of Trento.
References
Audus, L.J., 1972. Plant Growth Substances. Chemistry and Physiology,
vol. 1. Leonard Hill, London.
Blum, U., 2004. Fate of phenolic allelochemicals in soils—the role of soils
and rhizosphere microrganisms. In: Macı`as, F.A., Galindo, J.C.G.,
Molinillo, J.M.G., Cutler, H.G. (Eds.), Allelopathy Chemistry and
Mode of Action of Allelochemicals. CRC Press, London, UK, pp. 57–
76.
Corre, M.D., Schnabel, R.R., Shaffer, J.A., 1999. Evaluation of soil
organic carbon under forests, cool-season and warm-season grasses in
the northeastern US. Soil Biol. Biochem. 31, 1531–1539.
Einhellig, F.A., 2004. Mode of allelochemical action of phenolic
compounds. In: Macı`as, F.A., Galindo, J.C.G., Molinillo, J.M.G.,
Cutler, H.G. (Eds.), Allelopathy Chemistry and Mode of Action of
Allelochemicals. CRC Press, London, UK, pp. 219–238.
FAO–ISSDS, 1999. World Reference Base For Soil Resources. Versione
Italiana. Istituto Sperimentale per lo Studio e la Difesa del Suolo, Firenze.
Fierer, N., Schimel, J.P., Cates, R.G., Zou, J., 2001. The influence of
balsam poplar tannin fractions on carbon and nitrogen dynamics in
Alaskan taiga floodplain soils. Soil Biol. Biochem. 33, 1827–1839.
Gafta, D., 1994. Tipologia, Sinecologia e Sincorologia delle Abetine nelle
Alpi del Trentino. Camerino, Italy. Braun-Blanquetia, 12.
Gibbons, J.D., 1976. Non-Parametric Methods for Quantitative Analysis.
Holt Rinehart and Winston, New York.
Hammad, Y., Nalin, R., Marechal, J., Fiasson, K., Pepin, R., Berry,
A.M., Normand, P., Domenach, A.M., 2003. A possible role for
phenyl acetic acid (PAA) on Alnus glutinosa nodulation by Frankia.
Plant Soil 254, 193–205.
Harrison, A.F., Harkness, D.D., Rowland, A.P., Garnett, J.S., Bacon,
P.J., 2000. Annual carbon and nitrogen fluxes in soils along the
European forest transect, determined using
14
C-bomb. In: Schulze,
E.D. (Ed.), Carbon and Nitrogen Cycling in European Forest
Ecosystems. Max-Planck-Institut fu¨r Biogeochemie, Jena, Germany,
pp. 241–247.
Hartley, R.D., Buchan, H., 1979. High-performance liquid chromatogra-
phy of phenolic acids and aldehydes derived from plants or from the
decomposition of organic matter in soil. J. Chromatogr. 180, 139–143.
Hongve, D., van Hees, P.A.W., Lundstrom, U.S., 2000. Dissolved
components in precipitation water percolated through forest litter.
Eur. J. Soil Sci. 51, 667–677.
Howard, P.J.A., Howard, D.M., Lowe, L.E., 1998. Effects of tree species
and soil physico-chemical conditions on the nature of soil organic
matter. Soil Biol. Biochem. 30, 285–297.
Inderjit, 1996. Plant phenolics in allelopathy. Bot. Rev. 62, 182–202.
Inderjit, Nilsen, E.T., 2003. Bioassays and field studies for allelopathy in
terrestrial plants: progress and problems. Crit. Rev. Plant Sci. 22, 221–
238.
Kaiser, K., Guggenberger, G., Haumaier, L., Zech, W., 2001. Seasonal
variations in the chemical composition of dissolved organic matter in
organic forest floor layer leachates of old-growth Scots pine (Pinus
sylvestris L.) and European beech (Fagus sylvaticae L.) stands in
northeastern Bavaria, Germany. Biogeochemistry 55, 103–143.
Kalbitz, K., Kaiser, K., 2003. Ecological aspects of dissolved organic
matter in soils. Geoderma 113, 177–178.
Kalbitz, K., Solinger, S., Park, J.H., Michalzik, B., Matzner, E., 2000.
Controls on the dynamics of dissolved organic matter in soils: a review.
Soil Sci. 165, 277–304.
Kalbitz, K., Schmerwitz, J., Schwesig, D., Matzner, E., 2003. Biodegra-
dation of soil-derived dissolved organic matter as related to its
properties. Geoderma 113, 273–291.
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
199
Kuiters, A.T., Mulder, W., 1993. Water soluble organic matter in forest
soils. 1. Complexing properties and implications for soil equilibria.
Plant Soil 152, 215–224.
Mallik, A.U., Pellissier, F., 2000. Effects of Vaccinium myrtillus on spruce
regeneration: testing the notion of coevolutionary significance of
allelopathy. J. Chem. Ecol. 26, 2197–2209.
Marschner, B., Kalbitz, K., 2003. Controls of bioavailability and
biodegradability of dissolved organic matter in soils. Geoderma 113,
211–235.
Mayer, H., 1969. Tannereiche Wa¨lder am Su¨dabfall der Mittleren
Ostalpen, vol. 2. Verlages Press, Mu¨nchen.
Michalzik, B., Matzner, E., 1999. Dynamics of dissolved organic nitrogen
and carbon in a Central European Norway spruce ecosystem. Eur. J.
Soil Sci. 50, 579–590.
Nardi, S., Pizzeghello, D., Muscolo, A., Vianello, A., 2002. Physiological
effects of humic substances in higher plants. Soil Biol. Biochem. 34,
1527–1537.
Park, J.H., Kalbitz, K., Matzner, E., 2002. Resource control on the
production of dissolved organic carbon and nitrogen in a deciduous
forest floor. Soil Biol. Biochem. 34, 1391.
Pecina, R., Bonn, G., Burtscher, E., Bobleter, O., 1984. High-performance
liquid chromatographic elution behaviour of alcohols, aldehydes,
ketones, organic acids and carbohydrates on a strong cation-exchange
stationary phase. J. Chromatogr. 287, 245–258.
Pedrotti, F., 1981. Carta della vegetazione del foglio di Trento. Centro
Nazionale Ricerche, Rome.
Pedrotti, F., 1982. Carta della vegetazione del foglio di Mezzolombardo.
Centro Nazionale Ricerche, Rome.
Pedrotti, F., 1994. Guida all’escursione della Societa` Italiana di Fitoso-
ciologia in Trentino. 1–5 Luglio 1994, Camerino.
Pizzeghello, D., Nicolini, G., Nardi, S., 2001. Hormone-like activity of
humic substances in Fagus sylvaticae forests. New Phytol. 151, 647–657.
Pizzeghello, D., Nicolini, G., Nardi, S., 2002. Hormone-like activities of
humic substances in different forest ecosystems. New Phytol. 155, 393–
402.
Qualls, R.G., 2000. Comparison of the behaviour of soluble organic and
inorganic nutrients in forest soils. Forest Ecol. Manag. 138, 29–50.
Qualls, R.G., Haines, B.L., Swank, W.T., Tyler, S.W., 2002. Retention of
soluble organic nutrients by a forested ecosystem. Biogeochemistry 61,
135–171.
Rao, C.R., 1964. The use and interpretation of principal component
analysis in applied research. SankhyaA
˚ 26, 329–358.
Smith, C.K., Munson, A.D., Coyea, M.R., 1998. Nitrogen and phospho-
rus release from humus and mineral soil under black spruce forests in
central Quebec. Soil Biol. Biochem. 30, 1491–1500.
Souminen, K., Kitunen, V., Smolander, A., 2003. Characteristics of
dissolved organic matter and phenolic compounds in forest soils under
silver birch (Betula pendula), Norway spruce (Picea abies) and Scots
pine (Pinus sylvestris). Eur. J. Soil Sci. 54, 287–293.
Strobel, B.W., Hansen, H.C.B., Borggaard, O.K., Andersen, M.K.,
Raulund-Rasmussen, K., 2001. Composition and reactivity of DOC
in forest floor soil solutions in relation to tree species and soil type.
Biogeochemistry 56, 1–26.
Walkley, A., Black, I.A., 1934. An examination of the Degtjareff method
for determining soil organic matter and a proposed modification of the
chromic acid titration method. Soil Sci. 37, 29–38.
van Hees, P.A.W., Jones, D.L., Finlay, R., Godbold, D.L., Lunstro¨m,
U.S., 2005. The carbon we do not see—the impact of low molecular
weight compounds on carbon dynamics and respiration in forest soils:
a review. Soil Biol. Biochem. 37, 1–13.
200
D. Pizzeghello et al. / Chemosphere 65 (2006) 190–200
Document Outline
Wyszukiwarka
Podobne podstrony:
Zwalczanie szkodników - chemicznie czy biologicznie, TOKSYKOLOGIA, szkodniki magazynowe, zwalczanie
1.Składniki chemiczne komórki, BIOLOGIA MOJE PRYWATNE
skład chemiczny organizmów, biologia
Chemical And Ballistic Properties Of Black Powder
Functional similarities between computer worms and biological pathogens
ŚRODKI CHEMICZNE I BIOLOGICZNE
MAKROELEMENTY, Matura, Biologia Matura, składniki chemiczne komórek
Guide to the properties and uses of detergents in biology and biochemistry
[38]QUERCETIN AND ITS DERIVATIVES CHEMICAL STRUCTURE AND BIOACTIVITY – A REVIEW
Konwencje biologiczne i chemiczne
druk skutki zagrożeń biologicznych i chemicznych
Physical and chemical character Nieznany
biologia, ZANIECZYSZCZENIA WODY2, Zanieczyszczenia wód, wprowadzone do wód naturalnych organizmy żyw
Biologia część III, Chemiczne składniki komurki
Biologia część III, Chemiczne składniki komurki
Kolokwium 2 - biologia - sciaga, ANALITYKA CHEMICZNA- UŁ, Rok I, BIOLOGIA, egzaminy
Biologia część III, Chemiczne składniki komurki-tłuszcze
MEDYCYNA KATASTROF-BOJOWE ŚRODKI CHEMICZNE I BIOLOGICZNE, Medycyna, Medycyna ratunkowa
więcej podobnych podstron