Original article

Effect of fertilizer application on Urtica dioica and its element

concentrations in a cut grassland
Vladimra Mllerov

a
a
, Michal Hejcman
a, *
, Pavla Hejcmanov

a
b
, Vil

em Pavl
a
a
Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences, Kamck a 1176, Prague 6, Suchdol, CZ-165 21, Czech Republic
b
Department of Animal Sciences and Food Processing, Faculty of Tropical AgriSciences, Czech University of Life Sciences, Kamck a 129, Prague 6, Suchdol,
CZ-165 21, Czech Republic
a r t i c l e i n f o
Article history:
Received 27 November 2013
Accepted 13 May 2014
Available online
Keywords:
Fertilization
Stinging or common nettle
Nitrogen
Phosphorus
Plant nutrition
Potassium
a b s t r a c t
Little is known about the effects of nutrient availability in cut grasslands on growth characteristics of
Urtica dioica and its aboveground chemical composition (N, P, K, Ca, Mg, Cu, Fe, Mn and Zn). The effects of
N, P and K application on the growth of U. dioica were studied over ve years in a Dactylis glomerata
grassland cut twice per year under unfertilized control, P, N, NP and NPK treatments (300, 80 and 200 kg
of N, P and K ha
1
per year).
Nitrogen application in the form of NH
4
NO
3
over ve years decreased the soil pH, while P and K
application increased P and K availability in the soil. Over ve years, cover of U. dioica increased from 1%
initially to 7, 9, 58, 83 and 99% in the control, P, N, NP and NPK treatments, respectively. Concentrations of
N, P and Ca in the aboveground biomass of U. dioica were very high in comparison to other species and
concentrations of Cu, Fe, Mn and Zn were comparable with other grassland species. N and P limitation of
U. dioica growth was expected if concentrations of N and P in the aboveground biomass were lower than
25 g N kg
1
and 4 g P kg
1
in the phenological stage of owering.
We concluded that two cuts per year are not sufcient to suppress expansion of U. dioica under high N,
P and K availability. This probably explains why U. dioica survive also in frequently cut intensive grass-
lands under adequately high nutrient supply.
2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Urtica dioica L. (stinging or common nettle) is a perennial herb
that prefers slightly acidic to alkaline soil that is moist and nutrient
rich (Ellenberg et al., 1991). The species can grow in conditions
ranging from moderately shaded woodlands and hedgerows to
open habitats such as oodplains, pastures and meadows (

Srtek
and Teckelmann, 1998; Taylor, 2009). Grime et al. (2007)
described U. dioica as a ruderal species having an established
competitor (C) strategy. It is a successful coloniser in grasslands and
ruderal sites due to rhizome fragments and stolons, which continue
to grow until the death of the aerial shoots in the autumn. Urtica
dioica is able to suppress the growth of other herbaceous plants and
often forms monospecic stands on nutrient-rich sites (Al-Mufti
et al., 1977;

Srtek, 1993; Grime et al., 2007). Herbage of U. dioica
possesses a high nutritive value and was frequently used as an
additive into human, poultry and pig foodstuff in the past. Fresh
plants are little grazed by livestock because of stinging hairs
covering the stems and leaves of the plant, but dry plants in the
form of hay are taken up by livestock without any disorders (Grime
et al., 2007).
Urtica dioica is known as a particularly nitrophilous species with
high N and P requirements preferring well N- and P-supplied soils
with good water availability (

Srtek, 1995; Hill et al., 2004; Taylor,

2009). Once established, U. dioica can spread vigorously and it is
very difcult to eradicate it.
Although many authors mention the high competitive ability of
U. dioica and its ability to create dense stands on nutrient-rich soils,
no information is available regarding how quickly cover develops
after an increase in nutrient availability. In addition, dense stands of
U. dioica are characteristic of unmanaged or infrequently cut
grasslands (Prach, 2008; Klaus et al., 2011) and it is not clear
whether they can also develop in regularly managed grasslands
such as hay meadows cut twice per year. In addition, although the
positive effect of high nutrient availability on the growth of U. dioica
is known (Pigott, 1971; Taylor, 2009), little is known about how
nutrient availability affects growth characteristics of U. dioica plants
and N, P, K, Ca, Mg, Cu, Fe, Mn and Zn concentrations in its biomass.
* Corresponding author.
E-mail address: hejcman@fzp.czu.cz (M. Hejcman).
Contents lists available at ScienceDirect
Acta Oecologica
j ournal homepage: www. el sevi er. com/ l ocat e/ act oec
http://dx.doi.org/10.1016/j.actao.2014.05.004
1146-609X/ 2014 Elsevier Masson SAS. All rights reserved.
Acta Oecologica 59 (2014) 1e6
In 2007, we established a grassland fertilization experiment on a
Dactylis glomerata- and Festuca arundinacea-dominated grassland
with sporadic occurrence of U. dioica. The grassland represented a
hay meadow regularly cut twice per year (Hejcman et al., 2012a).
The immediate response of plant species composition to N
application was recorded and was found to be different to the
response over the four years of the study period. Highly productive
grasses (D. glomerata, F. arundinacea and Phleum pratense) were
promoted by N application in 2008 and then retreated together
with legumes (Medicago sativa, Trifolium pratense and Trifolium
repens) in all N treatments where the expansion of perennial forbs
(Urtica dioica and Rumex obtusifolius) and annual weeds (Galinsoga
quadriradiata, Impatiens parviora, Lamium purpureum and Stellaria
media) was recorded. In 2010, Festuca rubra was the dominant grass
in the control and P treatment, and species richness was lowest in
all treatments with N application. Mean annual dry-matter yield
over all years was 3.5, 3.9, 5.8, 5.6 and 6.8 t ha
1
in the control, P, N,
NP and NPK treatments, respectively. Concentrations of N in the
aboveground biomass ranged from 20.0 to 28.7 g kg
1
in the P and
N treatments; concentrations of P ranged from 3.2 to 3.7 g kg
1
in
the N and P treatments; and concentrations of K ranged from 24.1
to 34.0 g kg
1
in the NP and NPK treatments. The N:P, N:K and K:P
ratios did not correctly indicate the nutrient limitation of biomass
production, which was primarily N-limited, and K-limitation was
only recorded for high production levels in treatments with N ap-
plications. We concluded that high N, P and K application rates can
very quickly and dramatically change species composition, biomass
production and its chemical properties in sown cut grasslands.
High N application rates can be detrimental for tall forage grasses
and can support the spread of weedy species.
During the ve years, massive expansionof U. dioicawas recorded
in treatments with N application. The present study was therefore
focused on U. dioica to investigate how the N, P and K fertilizer
application under regular cutting management over ve years
affected: 1) soil chemical properties; 2) cover and growth charac-
teristics of U. dioica and 3) concentrations of macro (N, P, K, Ca, Mg)
and 4) micro (Zn, Fe, Mn, Cu) elements in its aboveground biomass.
2. Materials and methods
2.1. Study site
The fertilizer experiment was set up near the village of Msec,
45 km northwest of Prague (50

12
0
24
00
N; 13

51
0
40
00
E) at an altitude
of 490 m a.s.l. The study site was a at, species-poor meadow (35
vascular plant species per 240 m
2
) with a mean annual precipita-
tion and temperature of 550 mm and 8

C, respectively. The soil
type at the study site was Pararendzina (syn. Calcic Leptosol)
developed on mesozoic Ca-rich sediments. The pH/H
2
O in the up-
per 10 cm soil layer before the start of the experiment was 6.4. The
concentrations of plant-available (Mehlich III) P, K, Ca and Mg were
0.152, 0.267, 1.688 and 0.171 g kg
1
, respectively, and the total
(Kjeldahl) Ncontent was 2.3 g kg
1
. D. glomerata (visually estimated
cover of 45%), Festuca arundinacea (12%), Phleum pratense (9%) and
Taraxacum sp. (8%) were the dominant species before the estab-
lishment of the experiment. The meadow had been regularly cut
two- or three-times per year and once per ve years fertilized with
farmyard manure before establishment of the experiment. The last
manure application was performed in 2005. Nomenclature of
species follows a local ora (Kubat et al., 2002).
2.2. Experimental design
The experiment was established in summer 2007 on the
meadow with occasional occurrence of U. dioica (cover ca. 1%). A
completely randomised block designwas used with the unfertilized
control, P, N, NP and NPK fertilizer treatments replicated four times
(20 monitoring plots altogether, for the aerial photograph of the
experiment see Strnad et al. (2012)). The area of each individual
monitoring plot was 4 m 3 m. The application rates for N, P and K
in each plot were 150 kg N ha
1
(NH
4
NO
3
), 40 kg P ha
1
(Ca(H
2
PO
4
)
2
) and 100 kg K ha
1
(KCl), respectively. The rst fertil-
izer application was performed on 19th August 2007. From 2008 to
2011, yearly fertilizer application were added at the beginning of
March and then after the rst cut in June. Therefore, the total
annual application of N, P and K was 300 kg N ha
1
, 80 kg P ha
1
and 200 kg K ha
1
. The high application rates of N, P and K were
used to avoid any N, P or K growth-limitations of the highly pro-
ductive plant species. The experimental plots were cut twice per
year e rst in late September 2007 and then in early June and late
August yearly (for exact cutting dates see Fig. 1). The sward was
mown by engine scythe (Stihl FS 450 with metal knife) leaving a
stubble height of approximately 5 cm.
2.3. Soil sampling and chemical analysis
In each plot, one representative soil sample composed of ve
subsamples fromupper 0e10 cmsoil layer was collected in October
2011. The soil samples were air-dried, sieved through a 2-mm sieve
and sent to the accredited national laboratory Eko-Lab

Zamberk
(www.ekolab.zamberk.cz) where all chemical analyses were per-
formed. Total Ncontent was determined in a LECOTruSpec analyser
(Leco Corporation, St. Joseph, MI, USA) through combustion under
an oxygen atmosphere at 950

C, using helium as a carrier. The
organic C content was determined by the oxidation of the soil
sample in a mixture of potassium dichromate solution and sul-
phuric acid at 135

C and then measured spectrophotometrically.
Plant-available K, P, Ca and Mg concentrations were extracted by
Mehlich III reagent (Mehlich, 1984) and then determined by ICP-
OES (Varian VistaPro, Mulgrave, Australia). The pH value was
determined in 0.01 M CaCl
2
solution in the ratio 1:5.
2.4. Cover and growth characteristics of U. dioica
Cover of U. dioica was visually estimated in percentages before
the harvest in June and August from 2008 to 2011. The rst cover
estimation was done in September 2007.
In the middle of August 2011, ten stems of U. dioica were
randomly selected in each plot where U. dioica was recorded and
the following growth characteristics were measured: 1) length of
the stem; 2) number of leaves on the main stem; 3) number of
leaves per the main stem and all branches together; 4) number of
nodes on the main stem; 5) frequency of stems with branching
from ten selected; and 6) stem density per m
2
.
2.5. Biomass chemical properties
In each plot with the presence of U. dioica, we collected one
representative sample of aboveground biomass of U. dioica during
the second cut in August 2011. We cut plants 5 cmabove the ground
and collected 0.5 kg of fresh weight biomass. Samples were then
dried at 80

C until no further weight loss occurred. Dry samples of
aboveground biomass were analysed for N, P, K, Ca, Mg, Zn, Fe, Mn
and Cu concentrations. Nitrogen concentration in the plant samples
was determined by the same method as in the case of soil samples.
To determine P, K, Ca, Mg, Fe, Cu, Mn and Zn concentrations,
biomass samples were dissolved in aqua regia (the mixture of HNO
3
and HCl in a volume ratio of 6:1) and concentrations of elements in
the solution were determined using ICP-OES. The crude bre (CF)
was measured by the Weende method (AOAC, 1984). Ash
V. Mllerov a et al. / Acta Oecologica 59 (2014) 1e6 2
represents the residue left after ashing at 550

C. All analyses were
performed in the accredited laboratory Eko-Lab

Zamberk (http://
www.ekolab.zamberk.cz).
2.6. Data analysis
All analyses were performed using STATISTICA 9.0 software
(Statsoft, Tulsa, OK, USA). All data were checked for homogeneity of
variance and normality (Levene and ShapiroeWilk tests). One-way
ANOVA followed by post-hoc comparison using the Tukey HSD test
was applied to soil and biomass chemical properties. Cover data in
individual sampling dates and growth characteristics did not meet
assumptions for the use of ANOVA and were thus evaluated by non-
parametric KruskaleWallis test followed by multiple comparisons
of mean ranks for the detection of signicant differences between
treatments.
3. Results
3.1. Soil chemical properties
After ve years of fertilizer application, no signicant effect of
fertilizer treatment on the content of organic C, total N, C:N ratio
and concentration of plant-available Mg in the upper 10 cm soil
layer could be found (Table 1). In contrast, there was a signicant
effect of fertilizer treatment on pH, plant-available P, K and Ca
concentrations. The soil reaction was strongly acid in treatments
with N application and acid in the control and P treatments. The
pH/CaCl
2
ranged from 4.41 to 5.25 in N and P treatments, respec-
tively (Table 1). The concentrations of P and K were positively
affected by P- and K-application and ranged from 0.13 in N to
0.45 g kg
1
in P treatment and from 0.11 in N and NP to 0.26 g kg
1
in NPK treatment, respectively.
3.2. Cover and growth characteristics of U. dioica
Cover of Urtica in both control and P treatment remained low(9
and 8% respectively) during the whole study period. While at the
time of sampling U. dioica was recorded in only two of four control
and one of four P treatment plots, it was present in all plots of the N,
NP and NPK treatments.
Cover of U. dioica was relatively stable up to 9% in the control and
P treatment during the experiment, but gradually increased over
time in N, NP and NPK treatments (Fig. 1). Calculated separately by
one-way ANOVA for each year, the effect of treatment was signi-
cant at both sampling dates in 2010 and 2011. In the last sampling
date (18th August 2011), cover of U. dioica was signicantly affected
by treatment (P 0.009) and ranged from 7.8% 14.8
(mean standard deviation) in the P treatment up to 98.8% 2.5 in
the NPK treatment. Control and P treatments were signicantly
different from N, NP and NPK treatments (post-hoc comparisons).
The variability in cover of U. dioica increased in all treatments
during the experiment.
With the exception of frequency of branched stems and stem
density, the effect of fertilizer treatment on all other studied growth
characteristics of U. dioica was signicant (see Table 2 for details).
The shortest (104.9 cm) stems were recorded in the control and the
tallest (136.1 cm) in the NPK treatment. Number of leaves on the
main stem and number of leaves per the main stem and all
branches together were higher in treatments with N application
Fig. 1. Cover of Urtica dioica under investigated treatments from the start of the experiment in September 2007 until the second cut in August 2011. Mean values and standard error
of the mean (SE) are presented. Differences between treatments within each sampling date were evaluated by KruskaleWallis test. n.s. e result of the analysis was not signicant, *
or ** e result was signicant at a 0.05 or 0.01 probability level, respectively. Treatments: unfertilized control, application of P, N, NP and NPK.
Table 1
Effect of fertilizer treatments (control, P, N, NP and NPK) on mean values of soil chemical properties in the upper 10 cmsoil layer in October 2011. Calculated by one-way ANOVA
and using the Tukey post-hoc test (a 0.05), treatments with the different letter were signicantly different. F-value and P-value are results of one-way ANOVA, signicant
values are faced in bold. values indicate standard errors of the means (SE).
Variable Treatment F-value P-value
Control P N NP NPK
pH/CaCl
2
5.22
a
0.4 5.25
a
0.2 4.41
b
0.3 4.45
b
0.2 4.44
b
0.2 10 <0.001
C
org.
(g kg
1
) 24.1 1.7 28.8 0.6 26.9 2.1 27.1 0.2 29.2 1.9 1.9 0.168
N
tot.
(g kg
1
) 2.90 0.1 3.13 0.2 3.04 0.2 3.05 0.1 3.16 0.2 0.4 0.79
C:N 8.31 0.2 9.24 0.1 8.85 0.1 8.87 0.1 9.23 0.1 1.2 0.36
P (g kg
1
) 0.18
b
0.03 0.45
a
0.03 0.13
b
0.01 0.36
a
0.01 0.35
a
0.03 32.7 <0.001
K (g kg
1
) 0.16
bc
0.01 0.20
ab
0.02 0.11
c
0.01 0.11
c
0.02 0.26
a
0.02 13.6 <0.001
Ca (g kg
1
) 1.74
ab
0.2 2.34
a
0.2 1.02
b
0.2 1.49
b
0.1 1.49
b
0.2 7.1 0.002
Mg (g kg
1
) 0.12 0.02 0.11 0.01 0.15 0.01 0.11 0.01 0.11 0.01 1.6 0.218
V. Mllerov a et al. / Acta Oecologica 59 (2014) 1e6 3
than in the control and P treatment. In the NP treatment, the
highest number of leaves on the main stem (23) together with the
highest number of leaves per main stem(30) for all treatments was
recorded. Number of nodes on the main stemwas the lowest (14) in
the control and the highest (16) in the NP treatment. The highest
frequency of stembranching was recorded in the NP treatment and
no branching was recorded in the P treatment.
3.3. Biomass chemical properties of U. dioica
No signicant effect of fertilizer treatment on concentrations of
N, K and Ca was recorded, but the concentrations of P and Mg were
signicantly affected by treatment (Table 3). Mean P concentration
ranged from 3.9 0.5 in the N treatment to 5.6 0.5 g kg
1
in the
NP treatment. Mean Mg concentrations were lower in treatments
without N application and ranged from 2.3 to 5.1 1.4 g kg
1
in P
and NP treatments, respectively.
There was a signicant effect of fertilizer treatment on N:P and
K:P ratios in the biomass (Table 3). Mean N:P ratio ranged from4.51
to 7.61 0.3 in the P and N treatments, respectively, and the K:P
ratio ranged from 3.65 0.7 to 5.98 0.3 in NP and control,
respectively. There was no signicant effect of treatment on N:K
ratio.
With the exception of Cu, the effect of treatment on concen-
trations of microelements was not signicant (Table 3). The mean
Cu concentrations in the biomass ranged from 9.5 to
12.2 0.9 mg kg
1
in P and NPK treatments, respectively. The effect
of treatment on content of CF and ash was not signicant.
4. Discussion
4.1. Soil chemical properties
Nitrogen fertilizer primarily decreased the soil pH similarly as
reported in other studies (https://www.agronomy.org/
publications/jeq/articles/39/2/541Belay et al., 2002; https://www.
agronomy.org/publications/jeq/articles/39/2/541Saleque et al.,
2004). This is because during oxidation following fertilizer appli-
cation, ammonia compounds can release H

ions, which are the

source of soil acidity (Magdoff et al., 1997). The concentration of
plant-available P in the upper soil layer was high for nutrition of
grassland species in all treatments, probably because of surface
application of the farmyard manure in the past. The optimum level
for productive grasslands ranges from 0.05 to 0.15 g P kg
1
(Bud n akov a et al., 2004) and the lowest P concentration
(0.13 g P kg
1
) was observed in the N treatment and was caused by
increased P extraction by increased herbage yield stimulated by N
application (Hejcman et al., 2012a). High concentration of plant-
available P in treatment with P application was caused by P accu-
mulation in the soil due to the higher amount of annually applied P
(80 kg P ha
1
) than annually extracted by harvested biomass
(21e36 kg P ha
1
; Hejcman et al., 2012a). Application of K fertilizer
signicantly increased K availability in the soil but the level of
Table 2
Mean values of growth characteristics of the last sampling date in investigated treatments (control, P, N, NP and NPK): Length/Stem: length of the stem (cm); Leaves/Stem:
number of leaves on the main stem; Leaves/plant: number of leaves per the main stemand all branches together; Nodes/Stem: number of nodes on the main stem; BranStem:
frequency of stems with branching (in %); and StemDens: stem density per m
2
. Differences between treatments were evaluated by KruskaleWallis test. According to the
multiple comparisons of mean ranks, treatments with the different letter were signicantly different. H-value and P-value are results of KruskaleWallis test, signicant values
are faced in bold. values standard errors of the means (SE).
Growth characteristics Treatment H-value P-value
Control P N NP NPK
Length/Stem 104.9
b
3.9 117.8
ab
5.3 107.8
b
2.5 112.6
b
3.5 136.1
a
3.6 27.4 <0.001
Leaves/Stem 19.4
b
0.8 19.4
ab
0.9 21.8
ab
0.5 23.0
a
0.6 21.7
ab
0.8 14.4 0.006
Leaves/Plant 20.2
b
0.8 19.4
b
1 22.7
b
0.9 30.5
a
1.9 22.5
b
1.0 24 <0.001
Nodes/Stem 14.0
c
0.4 14.8
abc
0.6 14.8
bc
0.3 16.2
a
0.3 16.0
ab
0.4 20.3 <0.001
BranStem 10.0 3.2 0 10.0 1.8 52.5 8.7 5.0 0.9 2.8 0.587
StemDens 232 34 368 284 54 252 46 336 38 2.9 0.572
Table 3
Mean concentrations of elements, N:P, N:K and K:P ratios, crude bre (CF) and ash contents in the aboveground biomass of Urtica dioica of the last sampling date in investigated
treatments (control, P, N, NP and NPK). Calculated by one-way ANOVA and using the Tukey post-hoc test (a 0.05), treatments with the different letter were signicantly
different. F-value and P-value are results of one-way ANOVA, signicant values are faced in bold. values indicate standard errors of the means (SE). Treatments: unfertilized
control, application of P, N, NP and NPK.
Variable Treatment F-value P-value
Control P N NP NPK
N (g kg
1
) 23.2 2.3 21.2 29.9 1.8 29.1 2.5 27.5 2.2 1.5 0.275
P (g kg
1
) 4.3
b
0.1 4.7
ab
3.9
b
0.2 5.6
a
0.3 4.7
ab
0.2 6.8 0.007
K (g kg
1
) 25.4 0.5 25.5 20.9 1.6 20.3 2.1 26.3 1.6 2.4 0.118
Ca (g kg
1
) 25.1 5.5 21.5 28.0 1.7 33.0 2.5 29.1 2.9 1.4 0.294
Mg (g kg
1
) 2.6
ab
0.2 2.3
ab
4.9
ab
0.5 5.1
a
0.7 2.8
b
0.2 5.5 0.013
N:P 5.46
ab
0.6 4.51
ab
7.61
a
0.2 5.24
b
0.4 5.99
ab
0.8 3.9 0.037
N:K 0.91 0.1 0.83 1.44 0.1 1.49 0.2 1.07 0.2 2.5 0.107
K:P 5.98
a
0.2 5.43
a
5.31
a
0.2 3.65
b
0.4 5.61
a
0.1 12.5 <0.001
Zn (mg kg
1
) 48.9 2.3 44.9 75.8 12 63.4 3.4 66.3 3.1 1.7 0.233
Fe (mg kg
1
) 94.2 7.8 88.2 135.9 19 91.1 7 85.8 11 2.5 0.108
Mn (mg kg
1
) 171.5 8.5 160.0 183.5 37 206.3 45 190.8 41 0.1 0.975
Cu (mg kg
1
) 9.9
ab
0.38 9.5
ab
12.2
a
0.4 11.4
ab
0.5 9.5
b
0.5 6.9 0.006
CF (g kg
1
) 279.4 35 266.4 257.2 17 293.0 8 293.6 11 1.1 0.4
Ash (g kg
1
) 134.5 12 117.0 127.3 7.5 138.3 6.1 144.3 4.6 1.4 0.304
V. Mllerov a et al. / Acta Oecologica 59 (2014) 1e6 4
plant-available K was only medium in all treatments according to
evaluation for productive grasslands (Bud n akov a et al., 2004). This
was probably because of a negative annual K balance due to
herbage removal even in the NPK treatment (21 kg K ha
1
;
Hejcman et al., 2012a). In addition, K is highly mobile in the soil and
can be easily leached into groundwater (Kayser and Isselstein,
2005). Availability of K in the control decreased over ve years of
the experiment because of continuous K removal by harvested
biomass and no K application. Generally, availability of K in the soil
can be decreased relatively quickly by herbage removal in contrast
to availability of P and Ca (Pavl et al., 2013).
4.2. Cover and growth characteristics of U. dioica
Urtica dioica responded positively primarily to the Napplication.
The main message of the study is that two cuts per year were not
sufcient to prevent expansion of U. dioica under high Napplication
rate and optimum P availability in the soil. In addition to N supply,
expansion of U. dioica was supported by P and K application
although P availability in the soil was optimal for the growth of the
desired forage species even without P application. A positive effect
of simultaneous N, P and K application on expansion of U. dioica was
clear from its almost 100% cover in the NPK treatment in August
2011 even though its initial cover was only 1% in September 2007.
Fertilizer application substantially increased regeneration of
U. dioica after the cut and this is in agreement with the experiment
by Mutikainen and Walls (1995). Urtica dioica thus requires not only
high N supply, but also high P and K availability in the soil to fully
express its growth potential and to suppress other grassland spe-
cies (see also Pigott, 1971). In treatments with N application, fast
and large scale expansion of U. dioica was enabled by its clonal
spread by rhizomes and stolons. According to Bassett et al. (1977),
annual spread by rhizomes and stolons can be up to the distance of
2.5 m from the mother plant. New rhizomes are produced from
older rhizomes or from stem bases in the late summer and autumn
(Greig-Smith, 1948). Highly productive grasses (D. glomerata, F.
arundinacea and P. pratense) were promoted by N application in
2008 and then retreated together with legumes (M. sativa, T. pra-
tense and T. repens) in all treatments with N application where the
expansion of perennial forbs (U. dioica and R. obtusifolius) and
annual weeds (G. quadriradiata, I. parviora, L. purpureum and S.
media) was recorded. At the end of the experiment, F. rubra become
the dominant grass in the control and P treatment. In 2008, U. dioica
spread particularly into vegetation free gaps after retreat of tall
grasses which suffered from lodging and consequent fungus dis-
eases in treatments with N application (Hejcman et al., 2012a). In
following years, spreading clones of U. dioica effectively shaded
surrounding vegetation and inhibited thus growth of other species.
U. dioica is thus not only an indicator of unmanaged grasslands as
recorded by Prach (2008), but can also spread into grasslands cut
twice per year through the soil disturbance and if the nutrient
availability is adequately high.
Plant height was positively affected by P application. This is
consistent with conclusions by Pigott and Taylor (1964) that N
addition did not result in increased plant height, whereas P addi-
tion with or without N addition resulted in vigorous growth. In our
experiment, the signicantly highest plants were recorded in the
NPK treatment. It seems that stem growth of U. dioica is supported
not only by high N and P supply, but also by high K supply.
There was the tendency for higher frequency of branching in the
NP treatment than in other treatments, but there was a high vari-
ability in data between individual plots. In the NPK treatment,
branching was less frequent than in the NP treatment probably
because of tall plants and high density of U. dioica stems and
therefore lowlight availability there. Although fertilizer application
signicantly affected some growth characteristics, differences be-
tween treatments were relatively small in contrast to cover data.
4.3. Concentrations macro- and micro- nutrients
Nitrogen concentrations in the aboveground biomass of U. dioica
were high in comparison to herbage from different grassland
communities, where N ranged from 7 to 17 g kg
1
(see Hejcman
et al., 2010). High N concentrations in the aboveground biomass
are thus a characteristic trait of this nitrophilous species. Although
the effect of treatment on the concentration of N in the biomass
was not signicant, there was a trend for a gradual decrease in N
concentration from the N to the NP and the NPK treatment. This
was probably due to a dilution effect (Jarrell and Beverly, 1981) as
plants of U. dioica were taller in the NPK treatment with a higher
proportion of N-poor stems and a lower proportion of N-rich leaves
than in other treatments with N application. The lowest concen-
tration of N in biomass in P treatment was given by the lowest N
availability there and this was consistent with results for the lowest
concentration of N in total aboveground biomass (Hejcman et al.,
2012a). Our data indicate that N limitation of aboveground
biomass production of U. dioica can be recorded if the concentration
of N in the aboveground biomass is lower than 25 g N kg
1
in the
phenological stage of owering.
Phosphorus concentrations in the aboveground biomass were
high in comparison to herbage from different grassland commu-
nities, where P range from 0.93 to 2.1 g kg
1
(see Hejcman et al.,
2010). In addition to high N concentration, extraordinarily high P
concentration in the aboveground biomass is a characteristic trait
of U. dioica. P concentration in the aboveground biomass below
4 g kg
1
was recorded only in the N treatment. Walker et al. (1952)
recorded decreased herbage P concentration in N treatment on
soils low in P and, in contrast, increased P concentration in N
treatment on soils rich in P. In our study, the reduction of P con-
centration in the N treatment indicates that the growth of U. dioica
was partly limited by P availability there, but the limitation was not
strong because P concentration in U. dioica biomass was above
3 g kg
1
. Phosphorus limitation of U. dioica in the N treatment was
also indicated by its lower cover at the last sampling date in com-
parison to the NP and NPK treatments. It seems that P is the
growth-limiting nutrient for U. dioica if the concentration is below
4 g kg
1
in the aboveground biomass in the phenological stage of
owering. Our results clearly demonstrate that U. dioica requires
not only high N availability, but also high P availability in the soil to
cover its high P requirements (Pigott, 1971; Taylor, 2009).
Urtica dioica is well known for its high Ca concentration in the
aboveground biomass (Thompson et al., 1997; Taylor, 2009). The
highest Ca concentrations in total herbage in our experiment were
recorded in N, NP and NPK treatments with its highest cover
(Hejcman et al., 2012a).
The concentrations of all microelements were higher in the
treatments with N application, but not signicantly except Cu. This
may be due to the decrease in soil pHafter Napplication as mobility
of Fe, Mn and Zn in soil is generally higher under low pH (Hejcman
et al., 2009, 2012b). In addition, the NH
4
cation can enhance the
uptake of micronutrients such as Fe, Mn andZn (Diatta andGrzebisz,
2006). The concentrations of all micronutrients in the biomass were
in the optimum range for plant and animal nutrition. The normal
levels of Zn, Fe and Mn in plants range from 20 to 100, 50e150 and
40e200 mg kg
1
, respectively (Mahler, 2004; Vank et al., 2007).
4.4. Nutrient ratios
The N:P ratio in the biomass was below 8 in all treatments
indicating an inherently low N:P ratio in the U. dioica biomass. In
V. Mllerov a et al. / Acta Oecologica 59 (2014) 1e6 5
low productive wet grassland species, the biomass can be limited
by N if the N:P ratio is below 14 (Koerselman and Meuleman, 1996;
Olde Ventering et al., 2003; Gsewell, 2004). It seems that the
typical N:P ratio for nitrophilous species like U. dioica is below 10.
Although N:P ratio was below 8 in the N, NP and NPK treatments,
the Nlimitation of the aboveground biomass production of U. dioica
would not be expected there, because of a high surplus of applied N
in comparison to removed N in harvested herbage (Hejcman et al.,
2012a). The N:P ratio in the aboveground biomass can thus hardly
be used for estimation of nutrient limitation in the case of nitro-
philous U. dioica.
According to Olde Venterink et al. (2003), the K:P ratio can
indicate P limitation or N and P co-limitation when the value is
higher than 3.4, or K limitation or N and K co-limitation if the ratio
is lower than 3.4.
The K:P ratio in the biomass of U. dioica was above 3.4 in all
treatments. K limitation of biomass production was not indicated
by the N:K ratio, which was lower than the critical value of 2.3 for K
limitation (Olde Venterink et al., 2003). Only a weak K limitation of
biomass production can be expected in any of the treatments,
because the clay soil is generally able to supply plants with a high
amount of K even without K application (Kayser and Isselstein,
2005; Klaus et al., 2013).
5. Conclusions
1) Five years of high level of N, P and K fertilizer application
decreased soil pH in treatments with N application and
increased P and K availability in treatments with P and K
application.
2) Concentrations of N, P and Ca in the herbage of U. dioica were
very high in comparison to other species. N and P limitation of
U. dioica growth can be expected if concentrations of N and P in
the aboveground biomass in the phenological stage of owering
are lower than 25 g N and 4 g P kg
1
, respectively.
3) Concentrations of Fe, Mn and Zn were slightly increased by N
application probably because of a decrease in soil pH affecting
Fe, Mn and Zn mobility in the soil.
4) Although it is generally believed that regular cutting manage-
ment is able to prevent spread of U. dioica into grasslands, we
concluded that two cuts per year are not sufcient to suppress
U. dioica under high N, P and K availability. This probably ex-
plains why U. dioica survive also in frequently cut intensive
grasslands under adequately high nutrient supply.
Acknowledgements
The research was funded by the Czech University of Life Sciences
Prague by project CIGA 20124205.
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