Applied Soil Ecology 59 (2012) 106–115
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Applied Soil Ecology
journal homepage: www.elsevier.com/locate/apsoil
Arbuscular mycorrhizal fungi and soil microbial communities
under contrasting fertilization of three medicinal plants
Szymon Zubek a,∗ , Anna M. Stefanowicz b , Janusz Błaszkowski c ,
Maria Niklińska d , Katarzyna Seidler-Łożykowska e
a
Laboratory of Mycology, Institute of Botany, Jagiellonian University, Lubicz 46, 31-512 Kraków, Poland
Department of Ecology, Institute of Botany, Polish Academy of Sciences, Lubicz 46, 31-512 Kraków, Poland
c
Department of Plant Protection, West Pomeranian University of Technology, Szczecin, Słowackiego 17, 71-434 Szczecin, Poland
d
Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland
e
Institute of Natural Fibres and Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznań, Poland
b
a r t i c l e
i n f o
Article history:
Received 3 November 2011
Received in revised form 23 April 2012
Accepted 25 April 2012
Keywords:
Arbuscular mycorrhiza
AMF species richness
Dark septate endophytes
Microbial biomass
Olpidium spp.
Soil respiration
a b s t r a c t
The effects on soil microorganisms of a three-year cultivation programme using mineral and manure
fertilization on three perennial medicinal plant species, lemon balm (Melissa officinalis L.), sage (Salvia
officinalis L.), and lavender (Lavandula angustifolia Mill.), were studied. Root endophyte colonization,
namely arbuscular mycorrhizal fungi (AMF), dark septate endophytes (DSE) and Olpidium spp., as well
as AMF species richness and abundance in the soil, soil respiration, microbial biomass, and the activity
and functional richness of culturable bacterial communities were assessed. In the mineral fertilization
treatment (MRL), phosphorus and potassium fertilizers were incorporated at the beginning of the experiment, while nitrogen fertilizer was applied throughout its course. In the manure fertilization treatment
(MAN), animal manure was applied once four years prior to soil and plant sampling. The soil under MRL
was characterized by a higher pH and P, N and Ca contents. AMF colonization of sage under MAN was
lower in comparison to MRL; however, no significant differences were found in the case of lemon balm
and lavender. The results of the Plantago lanceolata L. laboratory bioassay in respect of the infectivity
potential of the AMF present in the soils tested were in accordance with the trends obtained from mycorrhizal colonization assessments of the medicinal plant species. DSE and Olpidium spp. were also observed
in the roots subjected to analysis; however, they were not abundant. In the case of sage, DSE were more
frequent in MAN, while no significant differences were found in lemon balm and lavender. In contrast,
Olpidium spp. were more abundant in the sage and lavender from MRL. Moreover, the lemon balm roots
were devoid of these endophytes. The spores of 15 AMF species from 10 genera were isolated from trap
cultures established from the soils collected in the field. The species richness found in MRL was higher
than that in MAN. Moreover, four AMF species were detected exclusively in the trap cultures established
from the soils collected from under lemon balm, two were exclusive to sage and one to lavender. The soil
basal respiration was significantly affected by the plant species, but not by the fertilization systems and
this parameter was lowest under lemon balm and highest under lavender. The microbial biomass differed
significantly between management types only for lemon balm and was lower in MAN than in MRL. Bacterial activity was affected neither by plant species nor by fertilization type. Bacterial functional richness
was significantly influenced only by the fertilization type in interaction with the plant species and was
higher in MAN than in MRL for sage. Our results suggest that moderate application of mineral fertilizers
does not suppress, and sometimes enhances, AMF and other soil microorganisms. Moreover, the effects
of the fertilization type on soil microbial properties depend on the plant species being cultivated.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The increasing demand for high-quality herbal products
and the necessity of standardizing plant raw materials for
∗ Corresponding author. Tel.: +48 12 4241798; fax: +48 12 4230949.
E-mail address: [email protected] (S. Zubek).
0929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.apsoil.2012.04.008
pharmaceutical purposes entail the cultivation of many medicinal plant species (Seidler-Łożykowska, 2002; Senderski, 2007; van
Wyk and Wink, 2008). With a view to producing high yield and
quality in medicinal plant biomass, conventional and organic cultivation systems have been tested and utilized (Seidler-Łożykowska,
2002; Seidler-Łożykowska et al., 2005). In general, conventional
cultivation utilizes relatively high levels of external inputs such
as fertilizers and pesticides. In contrast, organic systems focus on
S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
the application of manure and compost, without the use of chemical fertilizers and synthetic pesticides (Raviv, 2010a, 2010b). In
the case of both systems, interdisciplinary studies are needed in
order to evaluate the effect of management system applied on both
plant production and soil microorganisms (Bending et al., 2004;
Gianinazzi et al., 2010; Raviv, 2010b; Vestberg et al., 2011).
Symbiotic associations between plant species and arbuscular
mycorrhizal fungi (AMF; Glomeromycota) are common in terrestrial ecosystems, including those of anthropogenic origin, such as
agroecosystems (Smith and Read, 2008). AMF stimulate growth,
improve pathogen, heavy metal and salinity resistance and influence the content of secondary metabolites in plants. They also play
an important role in the formation and stabilization of soil aggregates (reviewed by Smith and Read, 2008; Gianinazzi et al., 2010).
Moreover, AMF can facilitate crops through increasing the host
plant’s nutrient uptake and growth while suppressing weed species
which are non-mycorrhizal or do not benefit from fungal associations. Therefore, they represent potential alternatives to costly and
environmentally damaging herbicides (Cameron, 2010; Rinaudo
et al., 2010). In view of the aforementioned functions of these symbiotic soil microorganisms, their presence in agricultural soils can
be crucial to the maintenance of soil sustainability and plant production (Gosling et al., 2006; Gianinazzi et al., 2010; Raviv, 2010b).
Additionally, non-symbiotic soil fungi and bacteria also contribute
to the sustainability of arable soils. Studies including both AMF and
other soil microbes are thus of primary importance. It should be
stressed that soil microbial communities in agroecosystems are
influenced by numerous factors such as soil type, plant species
being cultivated, fertilization, pesticide use and/or ploughing. Studies on soil microbes which take into account the effects of biotic
and abiotic factors, and their interactions, are therefore of interest.
A three-year field experiment was set up in order to study the
influence of mineral versus manure fertilization on (i) the soil biological properties and (ii) the quality and quantity of the medicinal
plant material obtained. This paper presents the results relating to
the influence of these two types of fertilization on soil microbes
in the soil collected from under three perennial medicinal plant
species, Melissa officinalis L. (lemon balm), Salvia officinalis L. (sage),
and Lavandula angustifolia Mill. (lavender). Fungal root endophyte
colonization of the cultivated plants, the potential of AMF propagules to colonize roots, the AMF species richness, soil respiration,
and microbial biomass, as well as the activity and functional richness of the culturable bacterial communities were investigated. To
the best of our knowledge, the effects of the two fertilization systems on both AMF and other soil microbial properties have not been
studied to date in respect of the cultivation of high-value medicinal plants. As AMF diversity, the potential of AMF to colonize roots,
microbial biomass and soil enzyme activities are often found to
decrease in conventionally managed soils (Bending et al., 2004;
Entz et al., 2004; Oehl et al., 2004; Flie␤bach et al., 2007; Galván
et al., 2009; Moeskops et al., 2010), we hypothesized that AMF colonization rates and species richness, as well as other soil microbial
properties, would be higher in a manure fertilization treatment
than in a mineral. Moreover, as microbial communities are often
driven by plant root exudates of different quality and quantity
(Grayston et al., 1996), we also expected plant species and/or interaction between the fertilization system and plant species to have a
significant effect on soil microbes.
2. Materials and methods
2.1. Experimental design
The studies were conducted in an experimental field belonging to the Institute of Natural Fibres and Medicinal Plants, and
107
located in Poznań-Plewiska, at 52◦ 25 N, 16◦ 58 E. Eighteen plots,
10 m2 (2.15 m × 4.5 m) each, arranged in two complete separate
blocks, were established for the experiment on the same soil type
(luvisol). Between 2005 and 2007, directly before the experiment
was set up in 2008, they had been subjected to an identical threeyear crop rotation (2005 – Phacelia tanacetifolia, 2006 – Pisum
arvense, 2007 – Fagopyrum esculentum) and tillage scheme. In order
to maintain the high standards requisite for medicinal plant raw
materials, a moderate intensity of management, in line with good
agricultural practice, was applied. Thus no herbicide and fungicide inputs were incorporated in either of the blocks and manual
and manually operated weed disposal methods were used in both
cases. Two contrasting fertilization regimes, namely mineral and
manure, were applied in the blocks. In the case of the mineral fertilization treatment (MRL), three universal fertilizers (N/CaO/MgO,
60:6:8 kg ha−1 , respectively; P2 O5 – 70 kg ha−1 ; K2 O – 100 kg ha−1 )
were applied, at the dosages recommended by producers, in the
spring of 2008. In the case of the nitrogen fertilizer, half of the
dosage was applied in the spring and the second half after the first
cut of the herb. Similarly, two applications of this fertilizer were
given, in the spring and after first cut of herb, throughout the course
of the experiment (2009–2010). In the manure fertilization treatment (MAN), animal manure (25,000 kg ha−1 ) was utilized once
four years prior to soil and plant sampling (2006). The amount of
manure incorporated was the medium quantity recommended for
medicinal plant species. It is suggested that lemon balm and sage be
cultivated in the second year after manure application, while lavender may be cultivated directly after it has been applied (Dachler and
Pelzmann, 1999; Kołodziej, 2010). No additional dosages of manure
were applied during the experiment in order to avoid microbiological contamination of the medicinal plant raw materials. No
chemical fertilization was used in MAN.
Three perennial plant species of high medicinal value, included
in the European Pharmacopoeia (2008) and cultivated for pharmaceutical purposes, were used in the experiment, namely a breeding
strain of lemon balm (Melissa officinalis L.), sage cvar. “Bona” (Salvia
officinalis L.), and a lavender population (Lavandula angustifolia Mill.
=L. officinalis Chaix); Lamiaceae. The plants names follow Mirek
et al. (2002), apart from S. officinalis, which follows Tutin et al.
(1964–1980). The lemon balm and sage seeds originated from
maintenance breeding and the lavender seeds from the collection of
the Institute of Natural Fibres and Medicinal Plants, Poznań. Earlier
investigations have indicated that these plant species are colonized
by AMF (Wang and Qiu, 2006; Zubek and Błaszkowski, 2009).
The lemon balm, sage and lavender seedlings, which had previously been grown in a greenhouse, were planted in the spring of
2008, in MRL and MAN, on 3 randomly chosen plots. On each of
3 replicate plots, 70 individual seedlings of a given species were
planted in a space of 0.45 m × 0.30 m. Herb cuts were performed
equally on MRL and MAN and on an annual basis, in order to
obtain the material for the assessments of yield and medicinal plant
quality.
2.2. Field sampling
The material for the analyses was collected in the third year of
cultivation. The plants were harvested during the flowering and
early seed formation period, on 29th June 2010. Root systems, with
soil, were excavated intact to a depth of ca. 20 cm and transported to
the laboratory in open plastic bags. Five subsamples were collected
from each of the 18 plots (2 fertilization types × 3 plant species × 3
replicate plots). Thus, 15 samples of each plant species from each
treatment were collected, giving a total of 90 samples. The roots
were stained for the visualization of fungal endophytes (see Section 2.5). The soil was used for chemical and microbial activity
analyses and was also utilized in a laboratory experiment aimed at
108
S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
determining the colonization potential of AMF propagules. Soil subsamples for the measurements of respiration, microbial biomass,
bacterial activity and richness were sieved (2 mm mesh) and kept
moist at 4 ◦ C until the analyses were carried out.
2.3. Chemical analyses of the soil
The soil dry weight was determined for all ninety samples after
drying at 105 ◦ C for 12 h. The water holding capacity (WHC) was
evaluated by means of the gravimetric method. In the case of other
analyses, the soil subsamples excavated at each plot were mixed
and analysed as a bulk sample. The total phosphorus content was
determined in an ammonium lactate extraction conducted in accordance with the Egner–Rim method, total nitrogen by means of the
Kjeldahl method, and total carbon using the Tiurin method (Mocek
and Drzymała, 2010). Exchangeable cations were measured with
a flame photometer and spectrophotometer in ammonium acetate
(Mocek and Drzymała, 2010).
2.4. Estimation of the AMF colonization potential
In order to determine the colonization potential of AMF propagules, namely the spores, mycelium in roots and extraradical
mycelium, which were present in the soils tested, a laboratory
experiment was conducted using Plantago lanceolata L. as the host
plant. For this purpose, 5 soil subsamples collected from under
the plants from each replicate plot were mixed to form one bulk
sample which was placed in two 500 ml pots. Five one-week
old P. lanceolata seedlings, which had been germinated on sterile sand, were planted in each pot. The pots were kept in sealed
Sigma–Aldrich sunbags under greenhouse conditions at 22 ± 2 ◦ C.
The light regime was 100–110 ␮mol PAR photons m−2 s−1 , 12/12 h.
The pots were arranged in a completely random manner. The cultures were watered, using 50 ml of distilled water, once a week.
After six weeks of growth, the plants were harvested and the roots
were stained (see Section 2.5) for the visualization of the AMF
mycelium.
2.5. Determination of fungal root colonization
The roots of the medicinal plant species collected in the field
and the P. lanceolata roots from the laboratory experiment were
prepared in line with Phillips and Hayman’s (1970) method, with
modifications. After being washed in tap water, the roots were
cleared in 10% KOH for 24 h and subsequently rinsed in water. The
material was acidified in 5% lactic acid in water for 24 h, then stained
with 0.05% aniline blue in 80% lactic acid (72 h), and finally stored in
80% lactic acid. Thirty ca. 1-cm-long root fragments per individual
plant were mounted on slides in glycerol:lactic acid (4:1).
Fungal root colonization was determined using a Nikon Eclipse
80i light microscope with Nomarski interference contrast optics.
The method proposed by Trouvelot et al. (1986) was followed for
assessment of arbuscular mycorrhizal development. The parameters evaluated were mycorrhizal frequency (F), relative mycorrhizal
root length (M), and relative arbuscular richness (A). An estimate
of mycorrhizal frequency (F%) is given as the ratio between root
fragments colonized by AMF mycelium and the total number of
root fragments analysed. The relative mycorrhizal root length (M%)
is an estimate of the amount of root cortex that is colonized by
AMF relative to the whole root system. Arbuscule abundance (A%)
is an estimate of arbuscule richness across the entire root system
(Trouvelot et al., 1986). In the case of dark septate endophytes (DSE)
colonization, the estimated frequency of DSE mycelium occurrence
in roots (FDSE %) was calculated in the same way as that used for the
presence of AMF. Additionally, the frequency of the occurrence of
fungi from the genus Olpidium (FOlp %) was assessed.
2.6. AMF species richness
2.6.1. Establishment of the trap cultures
Fifteen trap cultures on MRL soils and 15 on MAN were established for each plant species. For the establishment of the trap
culture, 100 g of fresh soil was placed into 9 cm × 12.5 cm, 500 ml,
plastic pots containing autoclaved, commercially available, coarsegrained sand, the proportions being grains 1.0–10.0 mm in diam. –
80.50%; grains 0.1–1.0 mm in diam. – 17.28%; and grains < 0.1 mm
in diam. – 2.22%. P. lanceolata was used as the host plant.
2.6.2. Spore isolation and identification
Six months after the establishment of the trap cultures, AMF
spores were extracted using the wet sieving and decanting method
(Gerdemann and Nicolson, 1963). The morphological properties
of the spores and their subcellular structures were determined in
material mounted on a slide in a drop of polyvinyl alcohol/lactic
acid/glycerol (PVLG) and in a mixture of PVLG/Melzer’s reagent
(4:1, v/v) (Omar et al., 1979). Observation of AMF spore characteristics was performed using an Olympus BX51 light microscope.
The fungal species names follow Schüßler and Walker (2010). The
slides containing the isolated spores were deposited in the slide
collection of the Department of Plant Protection, West Pomeranian
University of Technology, Szczecin.
2.7. Measurements of soil respiration and microbial biomass
Soil basal respiration (BR) and microbial biomass (Cmic ) were
measured in 5 soil samples from each plot, giving 90 samples in
total. Prior to the analyses, they were adjusted to 50% WHC and
incubated in a climate chamber at 22 ◦ C and 70% humidity, for
7 days. Distilled water was added every second day in order to keep
the soil moist. BR and Cmic , determined by substrate-induced respiration (SIR), were measured in fresh soil subsamples adjusted to
50% WHC. The samples were incubated in gas-tight jars at 22 ◦ C, and
the CO2 evolved was absorbed in 0.2 M NaOH. The excess hydroxide
was titrated with 0.1 M HCl after the addition of BaCl2 and phenolphthalein as an indicator. After 3 BR measurements, glucose
monohydrate, at 10 mg g−1 dwt soil, was added to the soil samples
to determine the Cmic . SIR measurements were performed after 4 h
of incubation.
Biolog GN2 plates were used to study the activity and functional
richness of the culturable heterotrophic bacterial communities. The
plates contain 95 soil carbon substrates and tetrazolium violet as
the microbial activity indicator. Bacteria oxidize the substrates and
simultaneously reduce the tetrazolium dye, which turns purple.
Colour development in the wells reflects the microbial activity on
particular substrates. The first stage of the procedure was the shaking of 5 g dwt of fresh soil samples in 50 ml of 0.9% NaCl for 1 h.
The extracts were then diluted 10 times and 1 plate per sample
was inoculated with 100 ␮l inoculum per well. The plates were
incubated at 22 ◦ C, and the colour development (optical density,
OD) was measured spectrometrically using a BIO-TEK ␮Quant; at
590 nm, on a twice-daily basis for ca. 112 h.
2.8. Calculations and statistical analyses
Cmic was calculated in accordance with the equation: Cmic
(␮g g−1 ) = 40.04x + 0.37, where x is the respiration rate given in
␮l CO2 h−1 g−1 (Anderson and Domsch, 1978). BR for each plot was
expressed as a mean calculated across 5 replicates and 3 measurements, while Cmic was given as a mean of 5 replicates. The optical
2.6 ± 0.3
2.3 ± 0.2
138.7 ± 6.1
32.7 ± 2.3
1.8 ± 0.1 B
1.4 ± 0.4
14.7 ± 1.3 B
13.8 ± 2.4
6.8 ± 5.7
1.8 ± 1.0
294.9 ± 22.6
48.5 ± 7.0
32.3 ± 20.3
22.7 ± 1.5
21.0 ± 2.6 B
20.3 ± 2.6
11.0 ± 0.6
12.3 ± 0.5
1.4 ± 0.1
1.1 ± 0.1
0.8 ± 0.2
0.6 ± 0.1
7.2 ± 0.1
5.2 ± 0.5
7.7 ± 0.1
6.2 ± 0.1
Lav MRL
Lav MAN
0.07 ± 0
0.05 ± 0.01
2.8 ± 0.3
2.7 ± 0.3
96.7 ± 5.8
40.0 ± 0
1.5 ± 0.2 A
1.0 ± 0
15.7 ± 2.5 AB
17.2 ± 2.4
4.3 ± 0.6
2.2 ± 0.8
203.5 ± 30.7
70.7 ± 12.7
28.6 ± 0.4
15.5 ± 8.8
21.3 ± 1.7 AB
23.8 ± 4.1
10.5 ± 0.5
11.2 ± 0.8
1.4 ± 0
1.2 ± 0
0.8 ± 0
0.7 ± 0
6.4 ± 0.2
5.1 ± 0.2
7.2 ± 0.4
6.4 ± 0.3
Sal MRL
Sal MAN
0.08 ± 0
0.06 ± 0.01
2.4 ± 0.5
2.6 ± 0.3
15.6 ± 1.7 A
19.9 ± 1.5
4.0 ± 4.3
2.7 ± 1.5
22.7 ± 2.1 A
28.1 ± 0.9
11.5 ± 1.3
11.9 ± 1.7
Mg
Ca
*
101.3 ± 43.9
38.0 ± 2.0
*
1.6 ± 0.2 AB
1.0 ± 0
Na
K
MgO
CaO
P2 O5
*
168.9 ± 108.3
55.1 ± 1.6
K2 O
*
34.6 ± 9.0
23.4 ± 1.0
*
1.3 ± 0.3
1.2 ± 0.1
*
0.8 ± 0.1
0.7 ± 0
Mel MRL
Mel MAN
3.2.2. Other endophytes
Dark septate endophytes (DSE) were found in all the plant
species; however, they were not present in all the root samples.
The mean frequency of DSE occurrence in roots (FDSE ) was low
(Table 3). The DSE occurrence in root systems was influenced
by the fertilization system in interaction with the plant species
(Table 2). DSE were the most abundant in the roots of sage and
the mean frequency of DSE in this plant was significantly higher
in MAN than it was in MRL. No significant differences in the
*
0.06 ± 0.01
0.07 ± 0.02
3.2.1. Arbuscular mycorrhizal fungi
Arbuscular mycorrhizae were observed in all plant species and
root samples. The interaction between plant species and fertilization type was significant only in the case of mycorrhizal frequency
parameter (F) (Table 2). It was lower in sage from MAN in comparison to all the other treatments. A similar tendency, although
not statistically significant, was found in relative mycorrhizal root
length (M) and relative arbuscular richness (A) (Fig. 1). The M and
A parameters were influenced by both plant species and fertilization type. The mean values of these mycorrhizal parameters in sage
were generally the lowest of all the plant species. In the case of
fertilization type, the mean values of M and A were lower in MAN.
*
6.6 ± 0.6
4.8 ± 0.1
3.2. Fungal root colonization of medicinal plants
*
7.2 ± 0.4
6.1 ± 0
The chemical properties of the soils from particular treatments
are reported in Table 1. The MAN soil was generally characterized by
a lower pH, organic matter content and the total and exchangeable
contents of elements in comparison to MRL. Statistically significant
differences were found between MRL and MAN in all the chemical
properties tested, apart from C/N, K2 O, MgO, K, and Mg. No statistically significant differences were found between plant species
with the exception of lemon balm and lavender (K2 O and K) and
sage and lavender (Na) (Tables 1 and 2).
Exchangeable cations
mg 100 g−1 of dry soil
3.1. Soil chemical properties
Total content
mg 100 g−1 of dry soil
3. Results
C/N
where vi is absorbance at time ti . The AUC was set to zero for
each well exhibiting a net OD < 0.1 at the end of the incubation
time. Bacterial activity was expressed as average area under curve
(AAUC). The functional richness of the bacterial communities (S )
was expressed as the number of substrates utilized on each plate.
Statistical analyses of the data on fungal root endophyte and
microbiological parameters were performed on the mean values obtained from subsamples in the case of each repetition plot
(N = 3). The data were evaluated using a two-way analysis of variance (plant species and type of fertilization). The significance of
differences between the treatments was tested following Tukey
(P < 0.05). Differences in AMF species richness between MRL and
MAN were tested with the non-parametric Mann–Whitney U test,
while differences in AMF species composition were analyzed using
the chi-squared test (P < 0.05). The analyses were carried out using
Statsoft’s STATISTICA ver. 9.
Organic matter %
x(ti − ti−1 ),
C%
2
N%
i=1
pH (KCl)
n
vi + vi−1
pH (H2 O)
AUC =
109
Treatment
density of a control Biolog well was subtracted from all substratecontaining wells, giving net ODs. All negative net ODs were set
to zero. Additionally, the ODs of the first measurement were subtracted from the ODs of all the subsequent measurements. Bacterial
activity on each substrate was expressed as area under curve (AUC),
calculated using the trapezoid method (Guckert et al., 1996) in line
with the equation:
Table 1
The chemical properties of the soil (mean ± SD; N = 3) collected from the experimental plots under two fertilization regimes (mineral – MRL, manure – MAN) of three medicinal plant species: Mel – Melissa officinalis, Sal – Salvia
officinalis, Lav – Lavandula angustifolia. Within each parameter, the capital letters indicate the statistically significant main effect of plant species; levels not connected by the same letter indicate statistically significant differences;
the asterisk (*) indicates the significant main effect of the fertilization type; for each P < 0.05. See Table 2 for details on main effects and interactions.
S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
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S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
Table 2
Results of two-way ANOVA for the effects of fertilization type, plant species and their interaction on the chemical and microbiological variables measured in the field
experiment; the effects in bold are significant at P < 0.05.
Soil property/Fungal root colonization
Chemical properties
pH (H2 O)
pH (KCl)
N
C
Organic matter
C/N
Total content
K2 O
P2 O5
CaO
MgO
Exchangeable cations
K
Na
Ca
Mg
Microbiological properties
Medicinal plants – fungal root colonization
F – mycorrhizal frequency
M – relative mycorrhizal root length
A – relative arbuscular richness
FDSE – frequency of DSE occurrence
FOlp – frequency of Olpidium occurrence
Laboratory bioassay – mycorrhizal parameters
F
M
A
Basal respiration (BR)
Microbial biomass (Cmic )
Bacterial activity
Bacterial functional richness (number of substrates utilized)
Fertilization type
Plant species
Fertilization type × Plant species
F1,12 = 65.22; P < 0.001
F1,12 = 94.73; P < 0.001
F1,12 = 7.63; P = 0.017
F1,12 = 25.56; P < 0.001
F1,12 = 16.65; P = 0.002
F1,12 = 3.05; P = 0.106
F2,12 = 1.57; P = 0.247
F2,12 = 3.78; P = 0.053
F2,12 = 0.82; P = 0.465
F2,12 = 0.89; P = 0.434
F2,12 = 0.66; P = 0.536
F2,12 = 1.34; P = 0.297
F2,12 = 2.62; P = 0.114
F2,12 = 1.37; P = 0.291
F2,12 = 2.09; P = 0.167
F2,12 = 0.48; P = 0.631
F2,12 = 0.93; P = 0.420
F2,12 = 0.35; P = 0.708
F1,12 = 3.90; P = 0.072
F1,12 = 5.94; P = 0.031
F1,12 = 54.39; P < 0.001
F1,12 = 3.84; P = 0.074
F2,12 = 5.22; P = 0.023
F2,12 = 0.83; P = 0.460
F2,12 = 2.42; P = 0.131
F2,12 = 0.23; P = 0.796
F2,12 = 2.17; P = 0.157
F2,12 = 0.05; P = 0.953
F2,12 = 3.45; P = 0.065
F2,12 = 0.59; P = 0.570
F1,12 = 2.73; P = 0.124
F1,12 = 22.16; P < 0.001
F1,12 = 76.26; P < 0.001
F1,12 = 0.17; P = 0.685
F2,12 = 4.37; P = 0.037
F2,12 = 4.87; P = 0.028
F2,12 = 1.67; P = 0.230
F2,12 = 1.15; P = 0.348
F2,12 = 2.41; P = 0.132
F2,12 = 0.16; P = 0.854
F2,12 = 3.21; P = 0.076
F2,12 = 0.82; P = 0.464
F1,12 = 28.29; P < 0.001
F1,12 = 9.70; P = 0.009
F1,12 = 4.76; P = 0.049
F1,12 = 6.29; P = 0.027
F1,12 = 79.39; P < 0.001
F2,12 = 18.54; P < 0.001
F2,12 = 4.25; P = 0.040
F2,12 = 4.34; P = 0.038
F2,12 = 11.16; P = 0.002
F2,12 = 44.70; P < 0.001
F2,12 = 7.24; P = 0.001
F2,12 = 3.14; P = 0.080
F2,12 = 2.50; P = 0.123
F2,12 = 4.10; P = 0.026
F2,12 = 28.24; P < 0.001
F1,12 = 6.30; P = 0.027
F1,12 = 7.65; P = 0.017
F1,12 = 2.03; P = 0.181
F1,12 = 2.09; P = 0.174
F1,12 = 0.53; P = 0.478
F1,12 = 1.16; P = 0.303
F1,12 = 1.88; P = 0.195
F2,12 = 1.25; P = 0.321
F2,12 = 0.50; P = 0.619
F2,12 = 0.34; P = 0.715
F2,12 = 53.48; P < 0.001
F2,12 = 23.34; P < 0.001
F2,12 = 2.17; P = 0.157
F2,12 = 2.61; P = 0.115
F2,12 = 3.75; P = 0.054
F2,12 = 4.78; P = 0.030
F2,12 = 5.70; P = 0.018
F2,12 = 0.11; P = 0.896
F2,12 = 7.86; P = 0.007
F2,12 = 3.77; P = 0.054
F2,12 = 12.86; P = 0.001
FDSE between treatments were found in lemon balm and lavender (Table 3). The percentage of DSE root colonization was low in
all the plant species (data not shown). Only single hyphae, accompanied sporadically by sclerotia, were found in the outer cortex
and rhizodermis. The mycelium either stained with aniline blue
or remained brownish. Single DSE hyphae were also observed on
root surfaces, and, as with the interior, they were not abundantly
mycorrhizal colonization (%)
100
80
a
a
a
a
MRL
a
MAN
60
AB
b
*
B
*
A
AB
40
A
B
20
0
present. In the case of the old, probably dead roots, which were not
included in the fungal colonization assessments, DSE were more
abundant.
The sporangia of Olpidium spp., which were stained with aniline
blue, were sporadically found inside sage and lavender root epidermal cells, and were not detected in all the samples analyzed.
Lemon balm root systems were devoid of these endophytes. The
frequency of the occurrence of Olpidium spp. (FOlp ) was influenced
by the fertilization system in interaction with the plant species
(Table 2). In both sage and lavender roots, FOlp was significantly
higher in MRL than it was in MAN (Table 3). The mean percentage of root infection by these endophytes was generally low in
both plants (data not shown). However, in the case of single root
fragments, sporangia were present in up to 40% of root epidermal
cells.
Mel
Sal
F
Lav
Mel
Sal
M
Lav
Mel Sal
Lav
A
Fig. 1. The effect of the fertilization regimes (mineral – MRL: white bars, manure
– MAN: grey bars) and host plant species on the mycorrhizal colonization of the
Melissa officinalis (Mel), Salvia officinalis (Sal), and Lavandula angustifolia (Lav) collected from the field experiment. Mycorrhizal parameters (mean ± SD; N = 3): F –
mycorrhizal frequency, M – relative mycorrhizal root length, A – relative arbuscular
richness. Within each mycorrhizal parameter, the small letters above the bars indicate the statistically significant effect of the interaction between plant species and
fertilization type; the capital letters show the significant main effect of plant species;
levels not connected by the same letter indicate statistically significant differences;
the asterisk (*) indicates the significant main effect of the fertilization type; for each
P < 0.05. See Table 2 for details on main effects and interactions.
Table 3
The frequency of dark septate endophyte (FDSE %) and Olpidium spp. occurrence
(FOlp %) (mean ± SD; N = 3) in the roots of the medicinal plant species (Mel – Melissa
officinalis, Sal – Salvia officinalis, Lav – Lavandula angustifolia) collected from the
experimental plots under two fertilization regimes (mineral – MRL, manure – MAN).
Within each parameter, the small letters following the values indicate the statistically significant effect of interaction between plant species and fertilization type;
levels not connected by the same letter indicate statistically significant differences;
P < 0.05. See Table 2 for details on main effects and interactions.
Plant species
FDSE
FOlp
Mel MRL
Mel MAN
5.24 ± 2.86 a
5.15 ± 1.34 a
Sal MRL
Sal MAN
4.84 ± 5.49 a
14.82 ± 3.15 b
26.0 ± 3.17 a
2.89 ± 5.01 b
Lav MRL
Lav MAN
1.11 ± 1.92 a
2.01 ± 1.34 a
10.94 ± 2.63 a
0.45 ± 0.77 b
0
0
S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
111
Table 4
The frequency of AMF spore occurrence, isolated from trap cultures established from the soils related to the medicinal plant species (Melissa officinalis, Salvia officinalis, and
Lavandula angustifolia) collected from the experimental plots under two fertilization regimes (mineral – MRL, manure – MAN).
Family
Fungal species
M. officinalis
MRL
Ambisporaceae
Ambispora gerdemannii (S.L. Rose, B.A. Daniels & Trappe) C.
Walker, Vestberg & A. Schüßler
Archaeospora trappei (R.N. Ames & Linderman) J.B. Morton
& D. Redecker
Diversispora epigaea (B.A. Daniels & Trappe) C. Walker & A.
Schüßler.
Entrophospora infrequens (I.R. Hall) R.N. Ames & R.W.
Schneid.
Scutellospora pellucida (T.H. Nicolson & N.C. Schenck) C.
Walker & F.E. Sanders
Claroideoglomus claroideum (N.C. Schenck & G. S. Sm.) C.
Walker & A. Schüßler
Claroideoglomus walkeri (Błaszk. & C. Renker) C. Walker &
A. Schüßler
Glomus aureum Oehl & Sieverd.
Glomus versiforme (P. Karsten) S.M. Berch
Glomus sp. 234
Funneliformis caledonium (T.H. Nicolson & Gerd.) C. Walker
& A. Schüßler
Funneliformis constrictum (Trappe) C. Walker & A. Schüßler
Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker &
A. Schüßler
Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot)
C. Walker & A. Schüßler
Paraglomus majewskii Błaszk. & Kovács
Archaeosporaceae
Diversisporaceae
Entrophosporaceae
Gigasporaceae
Claroideoglomeraceae
Glomeraceae
Paraglomeraceae
S. officinalis
MAN
MRL
L. angustifolia
MAN
MRL
MAN
7
7
7
13
7
7
7
67
7
7
87
60
67
60
80
7
13
13
7
27
7
20
93
20
7
13
33
80
7
20
27
93
7
7
20
20
20
13
13
7
7
3.4. AMF species richness
Arbuscular mycorrhizae were observed in all the P. lanceolata
root samples. No other fungal endophytes were found in any of
the root materials analyzed. In the case of F parameter, only the
effect of fertilization was significant (Table 2). In this case, the lower
values of F were observed in MAN. The AMF colonization potential
was significantly influenced by the fertilization type in interaction
with the plant species in M and A (Table 2). Statistically significant
differences in M and A parameters were only found between MAN
and MRL in the case of soils collected from under sage. In this case,
the AMF in MAN proved to be the least effective in P. lanceolata root
colonization (Fig. 2).
In total, the spores of 15 AMF species were isolated from the trap
cultures established from the soils collected in the field (Table 4).
The spores of Claroideoglomus claroideum, Funneliformis constrictum, Funneliformis mosseae, and Paraglomus majewskii were present
in the trap cultures of all treatments. C. claroideum and F. mosseae
were most frequently detected. In contrast, Ambispora gerdemannii, Claroideoglomus walkeri, Diversispora epigaea, Entrophospora
infrequens, Funneliformis caledonium, Glomus versiforme, and Rhizophagus irregularis were only isolated from single treatments and
occurred with the lowest frequency (Table 4). A higher species richness was observed in MRL, as compared to MAN (U = 10.5; P = 0.007).
Statistically significant differences were also found in AMF species
composition between fertilization types and between plant species
(2 = 3911.3; P < 0.001). A. gerdemannii, C. walkeri, D. epigaea, E.
infrequens, F. caledonium, Glomus aureum, Glomus sp. 234, and R.
irregularis were only found in MRL, whereas Glomus versiforme was
isolated exclusively from MAN. Four AMF species were found exclusively in the trap cultures established from the soils of lemon balm,
as well as two from sage and one from lavender (Table 4).
mycorrhizal colonization (%)
3.3. AMF colonization potential
100
*
MRL
80
MAN
a
60
ab
ab
40
b
ab
ab
ab
20
0
ab
Mel
Sal
F
Lav
Mel
Sal
M
Lav
a
b
Mel Sal
ab
ab
Lav
A
Fig. 2. The effect of the fertilization regimes (mineral – MRL: white bars, manure –
MAN: grey bars) and the medicinal plant species under cultivation (Mel – Melissa
officinalis, Sal – Salvia officinalis, and Lav – Lavandula angustifolia) on the mycorrhizal
colonization of Plantago lanceolata grown in soils collected from the field experiment; mycorrhizal parameters (mean ± SD; N = 3): F – mycorrhizal frequency, M
– relative mycorrhizal root length, A – relative arbuscular richness. Within each
mycorrhizal parameter, the small letters above the bars indicate the statistically
significant effect of the interaction between plant species and fertilization type;
levels not connected by the same letter indicate statistically significant differences;
the asterisk (*) indicates the significant main effect of the fertilization type; for each
P < 0.05. See Table 2 for details on main effects and interactions.
3.5. Soil respiration, microbial biomass, bacterial activity and
richness
Basal respiration ranged from 22.8 to 38.4 ␮M CO2 g
organic matter−1 24 h−1 . The BR was significantly affected by
the plant species, but not by the fertilization (Table 2). This parameter was lowest under lemon balm and highest under lavender
(Fig. 3). The microbial biomass varied from 5.0 to 8.4 mg g organic
matter−1 . The Cmic was generally highest under sage and lowest
under lemon balm. The effect of fertilization type on the Cmic was
seen only in interaction with plant species (Table 2). It differed
significantly between fertilization types only under lemon balm
and was lower in MAN (Fig. 4). Bacterial activity, measured using
the Biolog method, varied from 54.4 to 67.6 and neither plant
species nor fertilization effects were found in the case of this
parameter (Table 2). In turn, bacterial functional richness, ranging
112
S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
100
MRL
C
MAN
number of substrates utilized
basaal respiration (μM C
CO2 g om-1 24h-1)
50
40
B
30
A
20
10
Mel
Sal
from 78 to 85.7 utilized substrates, was significantly influenced by
the fertilization type in interaction with the plant species (Table 2).
The functional richness was significantly higher under sage in
MAN than in MRL (Fig. 5).
4. Discussion
AMF colonization rates and the abundance of arbuscules are
usually higher in plants grown on organically managed soils than
they are for conventional cultivation (Bending et al., 2004; Entz
et al., 2004; Galván et al., 2009; Kahiluoto et al., 2009). A high
soil phosphorus content usually results in low AMF colonization
(Smith and Read, 2008). Moreover, long-term P fertilization, even
at low levels, can reduce mycorrhiza formation (Mäder et al., 2000;
Bending et al., 2004). For instance, Duan et al. (2010) found low
colonization levels in maize, soybean, and wheat grown on fertilized soils. Similarly, Entz et al. (2004) observed lower colonization
rates in flax from conventional treatments than from organic. In
the aforementioned studies, the differences in colonization levels
microbial biomass ((mg g om--1)
MRL
MAN
bc
bc c
8.0
b
6.0
ab
a
4.0
2.0
0.0
Mel
Sal
b
a
ab
a
60
40
20
Mel
Sal
Lav
Lav
Fig. 3. The effect of the fertilization regimes (mineral – MRL: white bars, manure –
MAN: grey bars) and the medicinal plant species under cultivation (Mel – Melissa
officinalis, Sal – Salvia officinalis, and Lav – Lavandula angustifolia) on soil basal
respiration (mean ± SD; N = 3); om – organic matter. The capital letters show the
significant main effect of plant species; levels not connected by the same letter
indicate statistically significant differences; P < 0.05. See Table 2 for details on main
effects and interactions.
10.0
MAN
80
0
0
MRL
b
ab
Lav
Fig. 4. The effect of the fertilization regimes (mineral – MRL: white bars, manure –
MAN: grey bars) and the medicinal plant species under cultivation (Mel – Melissa
officinalis, Sal – Salvia officinalis, and Lav – Lavandula angustifolia) on soil microbial
biomass (mean ± SD; N = 3); om – organic matter. The small letters above the bars
indicate the statistically significant effect of the interaction between plant species
and fertilization type; levels not connected by the same letter indicate statistically significant differences; P < 0.05. See Table 2 for details on main effects and
interactions.
Fig. 5. The effect of the fertilization regimes (mineral – MRL: white bars, manure –
MAN: grey bars) and the medicinal plant species under cultivation (Mel – Melissa
officinalis, Sal – Salvia officinalis, and Lav – Lavandula angustifolia) on a number of the
substrates utilized by bacteria on the Biolog plates (functional richness; mean ± SD;
N = 3). The small letters above the bars indicate the statistically significant effect of
the interaction between plant species and fertilization type; levels not connected
by the same letter indicate statistically significant differences; P < 0.05. See Table 2
for details on main effects and interactions.
were mainly explained by the lower P concentration in organically
managed soils. In our study, phosphorus fertilization was applied in
MRL; the increased P soil content was consequently observed relative to MAN. However, mycorrhizal parameters were not decreased
owing to the increased concentration of available phosphorus in the
soil. The mycorrhizal colonization levels of lemon balm and lavender from both management systems did not differ significantly.
Moreover, there was a tendency towards lower colonization rates in
sage from MAN. The trends observed were confirmed by the results
of the P. lanceolata bioassay in respect of the colonization potential
of the AMF present in the soils tested. The lack of differences in
the mycorrhizal parameters of the lemon balm and lavender roots
collected from both treatments was not surprising. High levels of
colonization in soils rich in available P and the apparent insensitivity of AMF colonization to the incorporation of P fertilizers have
also been reported (Hamel et al., 1994; Vosátka, 1995; Ryan and
Ash, 1999). Moreover, the use of other readily soluble fertilizers,
particularly nitrogen fertilizers, has similarly been found to have
a negative impact on AMF colonization in some cases (Liu et al.,
2000; Burrows and Pfleger, 2002; Treseder and Allen, 2002), though
not in others (Ryan and Ash, 1999; Jumpponen et al., 2005). The
effect of various crop species on AMF colonization and abundance
in soils has been observed in earlier studies (Vestberg et al., 2005).
However, we found a significant interaction of plant species and
fertilization type in the case of sage. One of the possible explanations might be that the abundant colonization of sage was too
costly under the conditions of this particular MAN and the plant
could thus have down-regulated the AMF presence in the roots
and soil by means of root exudates. On the other hand, the moderate P supplementation in MRL, which had no deteriorating effects
on AMF, enhanced plant performance; hence it was possible for
this plant species to maintain a higher level of AM colonization.
This interpretation seems to be supported by the medicinal plant
mass data. The shoot mass of lemon balm and sage was lower in
MAN than in MRL, with no significant differences found in lavender
(Seidler-Łożykowska et al., unpublished). However, to fully confirm
this interpretation, an experimental approach is needed, with the
aim of conducting a parallel evaluation of plant cost and the benefit
of the symbiosis on both soil types, as revealed by plant performance, as well as an analysis of the compounds produced by roots
and their impact on AMF development.
S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
The total number of AMF species found in our studies is in accordance with previous investigations, where comparable species
richness was found in agroecosystems (Franke-Snyder et al., 2001;
Jansa et al., 2002). However, this is much lower than the 35 species
identified by Oehl et al. (2004) from both soil and trap cultures,
and higher than the 4 found directly in field soil by Vestberg et al.
(2005). It should be pointed out that fungal species which are probably hard to culture might have not sporulated in our trap cultures
and the AMF richness may thus presumably be higher. C. claroideum
and F. mosseae were present in the trap cultures of all treatments
and were the most frequently detected fungi. The results compare
with previous studies, where either both species (Vestberg et al.,
2005), or F. mosseae alone (Oehl et al., 2003, 2004), appeared to
be generalists in arable soils in Europe. Studies on AMF in agricultural soils have revealed enhanced mycorrhizal species richness
in organic plots (Oehl et al., 2003, 2004). However, Franke-Snyder
et al. (2001) reported that 15 years of conventional and organic
soil management caused no differences among fungal communities studied both in a field and in trap cultures. Similarly, Galván
et al. (2009), using molecular tools, found no differences in species
richness in onion roots grown under both types of cultivation. In
this study, higher species richness was found in MRL, which was
probably due to the higher pH and Ca content. Literature data confirm the importance of soil pH in the symbiosis of plants with AMF.
In Koomen’s et al. (1987) studies, most AMF tested preferred a
near-neutral pH. Although Zubek et al. (2009) did not find a close
correlation between AMF species diversity and soil pH, they found
the highest diversity in calcareous substrata. In this research, seven
AMF species were detected exclusively in the trap cultures established from the soils collected from under particular medicinal
plant species. Although most AMF associate with a wide range of
hosts, a specificity or selectivity of some plant species for particular
fungal symbionts exists (Helgason et al., 2002; Wubet et al., 2006;
Smith and Read, 2008). For instance, Wubet et al. (2006) found
that two coexisting, indigenous, forest plant species were associated with distinct AMF communities. Similarly, AMF host specificity
was reported by Vestberg et al. (2005) in several crops. In contrast,
Franke-Snyder et al. (2001) found a homogeneity of AMF communities in plant/host combinations and different cultivation systems.
Nevertheless, the possibility that, in the case of AMF species found
only once in the cultures, their occurrence might have been determined not by plant identity, but their rarity, cannot be excluded.
Dark septate endophytes (DSE) are frequently encountered
root-inhabiting fungi accompanying AMF in numerous plant
species, including those of medicinal importance (Fuchs and
Haselwandter, 2004; Zubek and Błaszkowski, 2009; Zubek et al.,
2011, 2012). The effects of these endophytes on plants are variable and may depend on the host species, fungal taxa or strains,
and environmental conditions (Jumpponen, 2001; Wu and Guo,
2008; Upson et al., 2009; Wu et al., 2010; Newsham, 2011). In
roots, DSE produce hyphae and sclerotia that cannot be regarded
as specialized interfaces for the transfer of nutrients between plant
and fungus. Therefore, these fungi probably do not influence plant
performance through direct contact with roots (Newsham, 2011).
Nevertheless, as suggested by Andrade-Linares et al. (2011) and
Mandyam et al. (2012), the effects of DSE on selected plant species
may be related to the intensity of their colonization of roots. Several mechanisms might explain the positive effects of these
fungi on plant growth, including enhanced protection from soil
pathogens, the synthesis of hormones or the mineralization of
organic compounds in soil by DSE (Mandyam and Jumpponen,
2005; Wu et al., 2010; Newsham, 2011). A meta-analysis published
recently by Newsham (2011) showed the positive role of DSE in
the stimulation of plant performance to have been particularly pronounced in soil conditions where nitrogen is present primarily in
organic form. The higher DSE frequency in sage from MAN may
113
suggest that owing to lower N soil content the plants may benefit
from DSE in these conditions. However, in order to reveal the nature
of DSE associations with the plants being investigated, research
under experimental conditions is necessary.
Several species from the Olpidium genus (Chytridiomycota) are
symptomless root parasites and are also the vectors of plant viruses
(Agrios, 2005; Webster and Weber, 2007). The sporangia of Olpidium spp. were found only sporadically inside sage and lavender
roots, and no symptoms of plant diseases were observed in the
plants under study. This suggests that these endophytes probably do not play an important role as pathogens in these plants.
The interpretation of the higher frequency of Olpidium spp. occurrence in the sage and lavender from MRL is, however, less apparent.
Nevertheless, as some Olpidium species transmit viruses that cause
diseases of agronomic significance (Verchot-Lubicz, 2003), the
monitoring of their presence in cultivated species would seem to
be essential and attention should be paid to the fact that they were
more frequently observed in the roots of the plants from MRL.
The influence of the fertilization type on the microbial parameters was relatively minor and its effects on the microbial biomass
and bacterial functional richness were observed only in interaction with the plant species. The microbial biomass was lower in
MAN than it was in MRL in the case of lemon balm, and did not
differ under the other plant species. On the other hand, the bacterial functional richness was higher in MAN than it was in MRL
only under sage. Numerous authors have found a positive effect
of organic farming on soil physicochemical and microbial properties (Gunapala and Scow, 1998; Bulluck et al., 2002; Melero et al.,
2006; Araújo et al., 2008; García-Ruiz et al., 2009). However, in most
studies organic amendments that improve soil physicochemical
properties and support microbial biomass such as animal manure
and/or composts, green manure, and rock phosphate were regularly applied. In the case of this study, animal manure was applied
only once, 4 years before the collection of the soil samples, while the
mineral fertilizers were used more regularly. Thus, such chemical
soil properties as organic matter and nutrient content were generally more beneficial to microorganisms in MRL than they were in
MAN. However, the positive response of the soil microbial parameters to the increased element content originating from the mineral
fertilization was not consistent. A potential explanation for this
inconsistency may be the fact that mineral fertilization enriches the
soil with nutrients but, contrary to organic amendments, it leads to
neither improvement of other soil properties such as aggregation,
water holding capacity, bulk density and CEC, nor to the inhibition of processes such as nitrate runoff, and, additionally, owing
to osmotic shock, may have a temporarily negative influence on
soil microorganisms (Drinkwater et al., 1995, 1998; Gunapala and
Scow, 1998; Bulluck et al., 2002). Because of the limited organic
fertilization in MAN and the potential diverse effects of mineral
fertilization on soil microorganisms, the fertilization effects on the
microbes in our study were neither strong nor unidirectional and
were highly dependent on the plant species.
Soil respiration and also microbial biomass were affected by the
plant species. Both parameters were lowest under lemon balm.
Respiration was highest under lavender and microbial biomass
under sage. It is well known that various plant species influence soil microbial communities differentially by supplying the
soil with diverse root exudates, as well as with plant litter of
different quality and quantity (Grayston et al., 1998; Marschner
et al., 2001; Reich et al., 2005). Additionally, essential oils containing active compounds are produced by many plants, in particular
those of medicinal importance (Barnes et al., 2007; Ponce et al.,
2003). For example, lemon balm contains at least 70 components
in volatile oils, predominantly monoterpenes and sesquiterpenes
(Barnes et al., 2007). In turn, more than 50 substances were identified in essential oils originating from sage (Santos-Gomes and
114
S. Zubek et al. / Applied Soil Ecology 59 (2012) 106–115
Fernandes-Ferreira, 2001; Barnes et al., 2007). The essential oils
produced by plants may possess a great potential to both inhibit
and enhance microbial activity and the effect depends on the plant
species (Vokou and Liotiri, 1999; Ponce et al., 2003).
The possibility that differences in respiration and microbial
biomass between plant species might have resulted from differences in the saprobic fungal component of the microbial
community cannot be excluded, as no effects of plant species on
bacterial activity and functional richness (Biolog analysis) were
detected. However, it is also possible that culturable bacteria growing on Biolog plates may not be representative for the entire
bacterial community and thus some bacterial differences may have
remained undetected.
In conclusion, our results suggest that, when moderate amounts
of mineral fertilizers are supplied, in comparison with a single addition of animal manure two years prior to growing medicinal plants,
mineral fertilization has no negative effects, and sometimes positive ones, on mycorrhizal fungal abundance and species diversity,
as well as on selected microbial parameters. Moreover, the effects
of the fertilization type on soil microbial properties may depend
on the plant species being cultivated. Combining the results relating to the impact of the fertilization on soil microorganisms and
those obtained from an ongoing analysis of plant raw materials
in the context of their medicinal usage, namely biomass, quality
and quantity of biologically active compounds, as well as chemical and microbiological contamination, will enable us to produce
some final conclusions and recommendations for farmers cultivating these herbs. Among the countries of Europe, Poland is one of the
leaders in the production of medicinal plant raw material for the
herbal industry (Seidler-Łożykowska et al., 2005; Pisulewska and
Janeczko, 2008). In this context, our studies may well be of particular importance for the development of effective methods for the
agricultural production of medicinal plants.
Acknowledgements
The authors would like to express their gratitude to the anonymous reviewers for their valuable comments on the manuscript.
The research received financial support from the Polish Ministry of
Science and Higher Education, under project No. N N304 381939
(2010–2013).
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