Journal of Arid Environments 73 (2009) 1117–1124

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Journal of Arid Environments

journal homepage: www.elsevier.com/locate/jaridenv

Comparison of soil bacterial communities associated with actinorhizal,

non-actinorhizal plants and the interspaces in the sclerophyllous matorral
from Central Chile in two different seasons
F. Farı́as, J. Orlando, L. Bravo, R. Guevara, M. Carú*
Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile,Casilla 653 Santiago, Chile

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

Article history: The spatial heterogeneity of resources in desert and semi-arid shrubland appears to be important in
Received 20 March 2008 determining higher soil bacteria abundance around plants than in soil without plant cover. Thus, these
Received in revised form bacterial communities could be important contributors to nutrient cycling in arid ecosystems. Bacterial
28 February 2009
diversity from Chilean sclerophyllous matorral was determined by Terminal Restriction Fragment Length
Accepted 11 June 2009
Polymorphism (T-RFLP). Soil samples associated with the actinorhizal plant Colletia hystrix, non-acti-
Available online 14 July 2009
norhizal plants and interspace soil without plant cover, were collected in May and October. The non-
actinorhizal and interspace soil differed significantly in their potassium content in May and pH in
Keywords:
Bacterial community October. The T-RFLP analysis revealed differences in the bacterial community structure from the different
Colletia hystrix habitats. The soil bacterial communities associated with plants were the most similar, whereas the
Sclerophyllous matorral interspace soil community differed in both sampling times. The factors that best explained the groupings
T-RFLP were potassium and pH. The greatest diversity was observed in the interspace soil. The Microbial
Community Analysis showed a significant proportion of T-RFs identified as Firmicutes and Proteobac-
teria. Likewise, spatial and temporal differences were observed in the main groups’ abundance. The
dominance of Firmicutes suggests that the sclerophyllous matorral could be a different ecosystem to
other arid and semi-arid soils with respect to the bacterial community structure.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction of exotic species resulting in an extensive loss of native vegetation

Desert and semi-arid shrubland ecosystems are characterized for
spatial heterogeneity of resources (Schlesinger et al., 1996; Ayarbe
and Kieft, 2000) and for its high vulnerability to anthropogenic
disturbances (Charney et al., 1975; Brown et al., 1997; Evans and
Belnap, 1999). According to Meigs (1953), 19% of the global land is
arid deserts and 14.6% is defined as semi-arid ecosystems. In Chile,
the arid and semi-arid regions constitute 22% of the total territory.
An example is the matorral of Central Chile, a shrubland plant
community composed of sclerophyllous (‘‘hard-leaved’’) shrubs and
small trees surrounded by grasses (Fuentes et al., 1984). It occupies
central Chile between 32 and 36 south latitude and is character-
ized by a temperate Mediterranean climate, with rainy winters and
dry summers (Gajardo, 1994). This ecosystem has been particularly
affected by degradation due to anthropogenic activities such as
logging, overgrazing, agriculture, urbanization and the introduction
* Corresponding author: M. Carú, Facultad de Ciencias, Universidad de Chile,
Casilla 653, Santiago, Chile. Tel.: 56-2-9787233; Fax: 56-2-272-7363.
E-mail addresses: mcaru@uchile.cl, margarita_caru@yahoo.com (M. Carú).
and soil erosion (Fuentes et al., 1986; Fuentes, 1990; Armesto et al.,
1995; Aronson et al., 1998). In fact, the matorral from central Chile
was once more extensive, but now exists as fragments in the coastal
ranges and Andean foothills. In the Andean range of central Chile,
some fragments are mainly formed by Colletia hystrix (Clos), a native
plant named actinorhizal for its ability to form a symbiotic associ-
ation with the nitrogen-fixing actinomycete Frankia (Silvester et al.,
1985; Carú and Cabello, 1999). The Colletia–Frankia symbiosis is an
important source of nitrogen input for the soil, in the form of
ammonia. These shrubs could play an important role as pioneer
plants in the colonization of nitrogen-poor soils or disturbed sites
facilitating the entry of new species after increasing the soil nitrogen
content similar to those suggested by other authors for similar plant
species (Becking, 1970; Dawson, 1986; Akkermans et al., 1989) and
giving rise to ‘‘fertility islands’’ (Binkley et al., 1982; Johnson, 1995;
González-Ruiz et al., 2008). Recently, Erickson et al. (2005) found
that the soil beneath another actinorhizal plant, Ceanothus cordu-
latus, had higher total nitrogen content than open canopy soils. In
addition, nitrate concentrations and net nitrogen mineralization
rates were greater in soils associated with C. cordulatus, than in
patches of non-actinorhizal plant species.

0140-1963/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jaridenv.2009.06.010
1118 F. Farı́as et al. / Journal of Arid Environments 73 (2009) 1117–1124
Some reports showed that differences in the quantity and/or
quality of nutrients with respect to soil without plant cover may
produce an increased microbial activity (Zak et al., 1994) and
changes in the microbial biomass (Kieft et al., 1998; Ayarbe and
Kieft, 2000), structure and genetic composition (Kuske et al., 2002)
of these microbial communities that lead to their differentiation.
Thus, soil bacteria abundance in dry soil environments would be
closely related to the accumulation of resources around individual
plants (Herman et al., 1995; Kuske et al., 2002) and these bacterial
communities could be important contributors to nutrient cycling in
arid land ecosystems. Although the evidence in other ecosystems
suggests that the microbial communities are critical for soil
productivity and stability (Yao et al., 2000; O’Donnell et al., 2001;
Grundmann, 2004; Bhatnagar and Bhatnagar, 2005), practically
nothing is known about the structure of natural soil microbial
communities from the vegetational patches of the matorral. This
study is focused on the soil bacterial communities associated with
a vegetational fragment from the Chilean sclerophyllous matorral.
Previous work showed that the soil bacterial community associated
with C. hystrix was different to bacterial assemblages from the
neighboring interspace soil (Orlando et al., 2007). However, it is
unknown if the structure and composition of the microbiota
associated with this actinorhizal plant differs from that associated
with the non-actinorhizal plant species found in the same area, and
which the edaphic factors could explain these differences. Similarly,
there is no information on the seasonal changes that these
communities could have in this Mediterranean ecosystem.
The objective of this work was to compare the soil bacterial
communities associated with plants and spaces between plants
(intespace), in a fragment from the sclerophyllous matorral of
central Chile in two different seasons. The study was carried out in
three habitats of the sclerophyllous matorral from the Andean
range: (i) soil associated with C. hystrix, (ii) soil associated with non-
actinorhizal plants and (iii) soil without plant cover (interspace soil).
We hypothesized that soil bacterial communities associated with
C. hystrix might be more similar between them than the soil bacterial
communities associated with non-actinorhizal plants and inter-
space soils. Besides we expected to find distinct communities in two
seasons (autumn and spring) due to the plant phenological changes.
The bacterial community profiles were determined by Terminal
Restriction Fragment Length Polymorphism (T-RFLP) (Liu et al.,
1997; Moeseneder et al., 2001) using 16S rDNA as molecular marker.
Numerous studies have previously shown T-RFLP to be both repro-
ducible and effective in characterizing bacterial communities in soil
(Dunbar et al., 2001; Kuske et al., 2002; Blackwood et al., 2003;
Yeager et al., 2005). Additionally, we determined the edaphic factors
of each habitat to evaluate if they could explain the differences
observed in the molecular profiles of the bacterial communities
studied.
2. Materials and Methods
2.1. Sampling Site
Soil samples were collected in the locality of ‘‘El Romeral’’, Cajón
del Maipo, Central Chile (33 48’S, 7014’W). The area studied is part
of the sclerophyllous matorral located in the Andean pre-mountain
range dominated by C. hystrix growing under natural conditions. In
addition to actinorhizal plants (C. hystrix), the selected area also
contains non-actinorhizal plants such as: Baccharis linearis, Quillaja
saponaria and Kageneckia oblonga.
The sampling was carried out during fall (May) and spring
(October) 2005 within a plot of 15  15 m. A total of nine soil
samples were collected from the soil associated with C. hystrix
(samples A1, A2 and A3), soil associated with non-actinorhizal
plants B. linearis, Q. saponaria and K. oblonga (samples NA1, NA2 and
NA3, respectively) and interspace soil (samples I1, I2 and I3). In
order to compensate the potential spatial variability associated
with the soil samples, 1 kg of soil was collected at each point. The
soil samples without plant cover were collected in the inter-canopy
spaces and the soil samples associated with plants were collected
10 cm from the plant stem, both at a depth of 0–10 cm. The soil
collected from each sampling site was homogenized, sieved and
one fraction from each sample was stored at -20  C for DNA
extraction and the rest was kept at 4  C in sterile plastic bags for
chemical analysis. Edaphic factors were determined as follows:
nitrogen by the Kjeldahl method, phosphorus by the molybdenum
blue method, potassium by flame photometry, soil organic matter
content by colorimetric determination following wet potassium
dichromate digestion and pH–H2O by potentiometric determina-
tion in a soil suspension in water (Forster, 1995).
2.2. Soil DNA Preparation
DNA was purified from 0.25 g of soil using the UltraClean Soil
DNA extraction kit (MoBio Laboratories, Solana Beach, CA, USA).
The DNA was resuspended in 50 mL of TE buffer (10 mM Tris-HCl
[pH 8.0], 1 mM EDTA) and stored at -20  C until analysis. The
concentration and quality of the DNA was determined electro-
phoretically in a 0.8% (w/v) agarose gel in TAE 1X buffer (40 mM
Tris-acetate, 1 mM EDTA [pH 8.0]) and visualized by ethidium
bromide staining (0.5 mg/ml). DNA was quantified using a size
standard of HindIII-digested lambda phage DNA (InvitrogenÒ).
2.3. PCR of 16S rDNA and T-RFLP Profiles
To amplify the 16S ribosomal DNA, primers fD1 50 -A GAG TTT GAT
CCT GGC TCA G-30 and rP2 50 -ACG GCT ACC TTG TTA CGA CTT-30
(Weisburg et al., 1991) were used. For the T-RFLP analysis, one of the
primers was labeled with 50 NED (2,70,80 -benzo-50 -fluoro-20 ,4,7-tri-
chloro-5-carboxyfluorescein) (Applied Biosystems, California, USA).
The fD1 and rP2 the 50 and 30 primers were marked to detect T-RFs,
respectively. For each reaction, the PCR mixture contained 2 mM
MgCl2, 120 mg/ml BSA, 120 mM of each dNTP, 0.2 mM of each primer
and 2.5 U of Taq polymerase (InvitrogenÒ) in PCR buffer in a final
volume of 25 ml to which was added between 10 and 20 ng of DNA.
The reactions were amplified in a GeneAmp PCR system thermal
cycler (Perkin Elmer). The program consisted of the following cycles:
initial denaturation at 94  C for 3 min, followed by 30 cycles of
denaturation at 94  C for 1 min, annealing at 57  C for 30 s, and
extension at 72  C for 2 min, with a final extension at 72  C for 7 min.
Six independent PCR reactions were performed and combined for
each soil sample. PCR products were purified with the UltraClean
PCR Clean-up kit (MoBio Laboratories, Solana Beach, CA, USA).
Amplified products were separated by electrophoresis on ethidium
bromide-stained agarose gels (1.2% w/v) to quantify the amplicon
and verify the correct size product.
The amplicon mixture was digested with 30 units of the restric-
tion enzyme HaeIII (InvitrogenÒ) for 3.5 h at 37  C, as recommended
by the manufacturer. The fragments were separated by electropho-
resis with an ABI-3100 DNA Genetic Analyzer (Applied Biosystems).
The length of the marked T-RFs was determined by comparison with
internal standards using the GeneScan 3.1.2 (ABI) program. Two
T-RFLP profiles were generated for each environmental sample, one
corresponding to the 30 T-RFs and the other to the 50 T-RFs.
2.4. Analysis of T-RFLP Profiles
T-RFs with a peak height greater than or equal to 30 fluores-
cence units were selected since they were clearly distinguishable
 F. Farı́as et al. / Journal of Arid Environments 73 (2009) 1117–1124
from the basal fluorescence. Reproducibility of patterns was
confirmed by repeated T-RFLP analysis using the same DNA
extracts. T-RFLP profiles from different samples were normalized as
a relative percentage of the total fluorescence units. Communities
were characterized by the number and height of the peaks. The
relative abundance of the T-RFs was expressed as a percentage. The
T-RFLP profiles were then transformed into a binary matrix
according to the presence (1) or absence (0) of each peak.
The similarity of samples, from binary data, was determined by
the Simple Matching coefficient (Sneath and Sokal, 1973) and the
dendrograms were constructed using the Unweighted Pairs Group
Method with Arithmetic Average (UPGMA) algorithm with boot-
strap values over 1000 replicates using the TREECON program (van
de Peer and de Wachter, 1994). A canonical correspondence anal-
ysis (CCA) was applied to evaluate the effect of the edaphic
parameters on the bacterial community diversity from the three
habitats studied (Ter Braak, 1986). The analysis was performed from
the binary matrix using the MultiVariate Statistical Package (MVSP)
version 3.12 h (GeoMem, Blairgowrie, UK).
For the identification of possible phylotypes present in the
sample, the MiCAÓ, Microbial Community Analysis 3 (http://mica.
ibest.uidaho.edu/trflp.php) was used which searches for coinci-
dences based on the T-RFs identified with the fluorescent labeled
primers and the Ribosomal Database Project II. The matches
obtained with the fluorescently marked fD1 primer were subse-
quently confirmed with the fluorescent marked rP2 primer.
2.5. Calculation of Diversity Indices
To evaluate the richness and evenness of each community, we
used the relative T-RF abundance data obtained using the fD1
primer to estimate the diversity of the 16S rRNA gene by calculating
the Shannon index (H0 ) according to the formula H0 ¼ -Spi ln pi
(Begon et al., 1990), where pi is the proportion of each T-RF from the
total number of T-RFs of each profile, and the evenness index (J)
using the formula J ¼ H/Hmáx (Begon et al., 1990), where Hmáx ¼ ln S,
where S is the number of T-RFs within each sample. These values
were calculated using the MVSP program version 3.12 h (GeoMem,
Blairgowrie, UK). Given that any T-RF fragment may represent
sequences from multiple phylogenetic groups, and may therefore,
not represent a true phylotype in the traditional sense, we will use
the number of T-RFs as a measure of the ‘‘bacterial richness’’
calculated as the total number of distinct T-RF sizes in each profile.
2.6. Statistical Analysis
Edaphic factors (soil organic matter, nitrogen, phosphorus,
potassium and pH) and diversity indices (Shannon index, equita-
bility and number of T-FRs) were treated statistically by two-way
analysis of variance (ANOVA) using the GraphPad Prism version 4.0
program (GraphPad Software, Inc.). Season and habitat were used
as factors. Pairwise multiple comparison between all treatment
means were made by using the Bonferroni’s test at P < 0.05.
3. Results
3.1. Edaphic Factors
In general, the edaphic factors did not show significant differ-
ences between habitats or between seasons. However, significant
differences were observed between the non-actinorhizal and
interspace samples, whereby these soils differed in their potassium
content in May (two-way ANOVA, habitat factor, F ¼ 3.78; P ¼ 0.05)
and pH in October (two-way ANOVA, habitat factor, F ¼ 4.84;
P ¼ 0.04). Similarly, the pH showed statistically significant seasonal
1119
difference between the non-actinorhizal soil samples (two-way
ANOVA, season factor, F ¼ 6.32; P ¼ 0.03) (Table 1). There was
a seasonal tendency whereby soils associated with plants (A and NA
samples) showed a greater N content in May than in October.
Likewise, the soils associated with C. hystrix (A samples) showed
a greater N content than soils associated with NA.
3.2. Bacterial Community Diversity
Each soil bacterial community was characterized by two T-RFLP
profiles generated by the 50 and 30 T-RFs of the 16S rDNA. The
number (richness) and relative abundance (evenness) of each T-RF
in the profiles were used to compare the bacterial communities. For
this analysis, the mean number of T-RFs per sample in the sampling
sites ranged from 25 to 33 in May and 21 to 65 in October. A greater
richness of T-RFs was observed in October in the interspace soil
samples with respect to the soils with plant cover (two-way
ANOVA, habitat factor, F ¼ 27.97; P < 0.0001). Additionally, the
interspace soil showed a greater number of T-RFs in October than in
May. The bacterial community profiles from the interspaces were
significantly affected by the habitat, season, and their interaction
(two-factor ANOVA; F ¼ 27.97; P < 0.0001; F ¼ 13.95; P ¼ 0.003;
F ¼ 26.21; P < 0.0001, respectively). In this case, habitat factor
explained 39% of the variance and season factor only 10% (Table 1).
The similarities between the three habitats (A, NA and I) in each
season, based on a distance matrix analysis of community T-RF
profiles, are illustrated in Fig. 1a and 1b. This analysis indicated that
the soil bacterial communities associated with plants (A and NA)
were the most similar, whereas the interspace soil (I) community
differed in both sampling times, where the differences were more
pronounced in the month of May. Each group showed a high boot-
strap value. Furthermore, the samples associated with actinorhizal
plants always grouped separately from the non-actinorhizal plants,
suggesting that the plant species also affects the T-RFLP profile.
In order to identify the environmental variables that were most
influential in determining the community diversity, we applied the
CCA analysis (Fig. 2). In May, the first two axes accounted for 56.0%
of the variance present in the samples (Fig. 2a) while in October,
they accounted for 57.5% of the variance (Fig. 2b). In May, the
samples from non-actinorhizal plants formed a well-defined
cluster; this result was unexpected because these soil samples
derived from different species of non-actinorhizal plants. However,
axis 1 clearly separated this cluster from the cluster formed by the
samples from C. hystrix and the interspaces (Fig 2a). In May, the
edaphic factors most correlated with axis 1 were potassium and
phosphorus with intra-set correlations of 0.69 and -0.59, respec-
tively. On the contrary, the two factors correlated to axis 2 were pH
(intra-set correlation of 0.75) and nitrogen (intra-set correlation of
-0.67) which separated the interspace soil samples from those
associated with plants (Fig. 2a). In October, the groupings were less
consistent than in May except for the interspace samples. These
results were explained again by potassium (intra-set correlation of
0.86) and to a lesser extent by phosphorus (intra-set correlation of
0.33). Conversely, axis 2 clearly separated the interspace soil
samples from those associated with non-actinorhizal plants, the
two factors more correlated to this axis were pH (intra-set corre-
lation of 0.88) and nitrogen (intra-set correlation of -0.78) (Fig. 2b).
3.3. Bacterial Community Composition
An approximation of the composition of the bacterial commu-
nities was obtained by applying the MiCA tools to the T-RFLP profiles.
The most represented T-RFs of those identified would correspond to
Firmicutes and Proteobacteria, including Alpha-, Beta-, Gamma- and
Delta-proteobacteria. As expected, according to the T-RF profiles,
1120 F. Farı́as et al. / Journal of Arid Environments 73 (2009) 1117–1124

Table 1
Comparison of the edaphic factors, diversity indices and T-RFs number  Standard errors for the bacterial communities associated with three soil habitats: A (soil associated
with C. hystrix), NA (soil associated with non-actinorhizal plants) and I (interspaces between plants); during two seasons: May and October.

Two-way Response
ANOVA
May October Season factor Bonferroni’s
H  S(1) test

A NA I A NA I F P A NA I
Edaphic pH 7.0  0.06(a) 7.1  0.07(a) 7.2  0.1(a) 7.2  0.05(ab) 7.5  0.06(a) 7.1  0.09(b) 6.32 0.03 ns * ns
factors F ¼ 4.84; 4.87 0.03
P ¼ 0.04
MO (%) 7.5  1.6(a) 5.3  0.6(a) 5.1  0.3(a) 7.6  1.2(a) 8.2  1.1(a) 7.0  1.2(a) 3.09 0.10 ns ns ns
F ¼ 0.82; 0.78 0.48
P ¼ 0.46
Nitrogen (ppm) 53  21.5(a) 30.7  18.7(a) 10.7  1.3(a) 28.0  1.5(a) 10.3  0.3(a) 35.3  11.3(a) 0.44 0.52 ns ns ns
F ¼ 1.49; 2.38 0.13
P ¼ 0.26
Phosphorus (ppm) 68.3  22.5(a) 51.3  3.2(a) 62.0  4.7(a) 78.2  15.8(a) 50  8.2(a) 45.8  10.9(a) 0.05 0.82 ns ns ns
F ¼ 1.85; 0.53 0.60
P ¼ 0.20
Potassium (ppm) 469  93(ab) 599  24(a) 348  14(b) 497  34(a) 540  163(a) 354  38(a) 0.01 0.90 ns ns ns
F ¼ 3.78; 0.16 0.85
P ¼ 0.05
Bacterial Diversity (H0 ) 2.99  0.18(a) 2.70  0.13(a) 3.07  0.18(a) 2.80  0.38(a) 2.86  0.19(a) 3.54  0.13(a) 0.68 0.43 ns ns ns
Diversity(2) F ¼ 3.26; 1.20 0.33
P ¼ 0.07
Equitability (J) 0.86  0.04(a) 0.85  0.02(a) 0.93  0.02(a) 0.83  0.11(a) 0.94  0.02(a) 0.85  0.03(a) 0.02 0.89 ns ns ns
F ¼ 0.66; 1.6 0.24
P ¼ 0.53
No. TRFs 33  2(a) 25  3(a) 28  6(a) 29  1(a) 21  3(a) 65  1(b) 13.95 0.003 ns ns ***
F ¼ 27.97 26.21 0.0001
P < 0.0001

Values following the same lowercase letter are not significantly different between habitats by two-way ANOVA following Bonferroni’s test (P < 0.05).
* P < 0.05; ***P < 0.001 by two-way ANOVA following Bonferroni’s test.
(H0 ) Shannon–Weaver index.
(1)
H: Habitat (A, NA, I); S: sampling season (May and October).
(2)
The bacterial diversity data are based on the fD1 primer analysis.

differences in the bacterial composition were observed in the
samples collected from different habitats during the same season.
For example, in May, Firmicutes dominated in the actinorhizal soil
samples collected while Proteobacteria were dominant in the non-
actinorhizal and interspace samples. Similarly, spatial and temporal
differences were observed with respect to the abundance of main
groups obtained (Fig. 3). The samples collected in October from soil
associated with C. hystrix showed a higher abundance of Alpha- and
Gammaproteobacteria than samples collected in May. In contrast,
the Firmicutes represented an abundance of almost 45% in May, but
less than 10% in October. However, the soil derived from the inter-
spaces did not show changes in the abundance of Firmicutes in both
sampling times. The Gamma-proteobacteria group was more
abundant in May in the interspace soil than in October, an inverse
effect was observed in the soil associated with actinorhizal plants.
In the case of the Alpha-proteobacteria, these showed seasonal
changes with respect to their abundance in the three habitats. In
general, groups such as Bacteroidetes, Acidobacteria, Cyanobacteria
and Actinobacteria were found in lower proportions, and smaller
seasonal changes in their abundance were observed. There is an
important portion of the soil microbial community that remains
unknown. This is reflected by the T-RFs from the three habitats
studied which could not be assigned to any bacterial group. This set
of undetermined T-RFs also showed important seasonal variations in
the bacterial communities from soil associated with non-actino-
rhizal plants and interspaces.
4. Discussion
In this study, we compare the soil bacterial communities from
three habitats associated with native sclerophyllous plants present
in the matorral from central Chile in autumn and spring. The T-RF
analysis was used at a field-scale to determine the diversity of the
16S rRNA genes and to evaluate differences among habitats and
seasonal changes. Currently, there is a lack of information on the
microbiology of these semi-arid soils from the matorral. A previous
study using T-RFLP analysis demonstrated that there are differences
between the structures of the bacterial community from soil asso-
ciated with C. hystrix compared to bulk soil (Orlando et al., 2007). The
approach based on the T-RFLP analysis of 16S rRNA genes to assess
the spatial or temporal heterogeneity of microbial communities at
the field scale has been used in different environments (Marsh,1999;
Dunbar et al., 2000; Sánchez et al., 2004; Morales et al., 2006;
Hartmann and Widmer, 2006; Hullar et al., 2006).
Our data showed that the environmental samples group
according to their origin, suggesting that the diversity character-
ized by the T-RFLP pattern of these bacterial communities (A, NA
and I), was defined by the habitat they occupy. In fact, various
studies have indicated that the plant species have an influence on
the structure and composition of the microbial communities
(Duineveld et al., 2001; Kuske et al., 2002; McCulley and Burke,
2004; Orlando et al., 2007). Likewise, in the absence of plant cover
the microbial community presents a structure different from that of
the community associated with plants in both sampling times.
According to the Shannon index, the bacterial communities of
the interspace soil showed a greater diversity than the soil associ-
ated with plants. This result could be unexpected since it has
generally been described that soils associated with plants show
higher values for total soil nutrients, microbial biomass, C, N, and
bulk measurements of microbial activity than in the interspaces
(Charley and West, 1977; Bolton et al., 1993). In fact, the hypothesis
that vegetational patches constitute islands of fertility in semi-arid
 F. Farı́as et al. / Journal of Arid Environments 73 (2009) 1117–1124 1121

a 2.0
pH

1.3

0.7

CCA2 - 26.8
0.0

-0.7 P

S.O.M. K
-1.3
N

-2.0
-2.0 -1.3 -0.7 0.0 0.7 1.3 2.0
CCA1 - 29.2
Vector scaling: 2.34 A NA I

b 2.7
pH
K
1.8

0.9 S.O.M.

CCA2 - 26.7
0.0
P
-0.9

-1.8
N

-2.7
-2.7 -1.8 -0.9 0.0 0.9 1.8 2.7
CCA1 - 30.8
Vector scaling: 2.68 A NA I

Fig. 2. Ordination diagram from the canonical correspondence analysis (CCA) of the soil
bacterial communities analyzed by T-RFLP for: (a) May and (b) October. A: actinorhizal,
Fig. 1. Dendrograms based on 50 T-RF and 30 T-RF profiles of 16S rDNA, showing simi-
NA: non- actinorhizal and I: interspace soil samples. The arrows indicate correlation
larity between the bacterial communities from actinorhizal (A), non-actinorhizal (NA)
coefficients between environmental variables and the given ordination axes.
and interspace (I) soil samples. (a) May, (b) October. The dendrograms were con-
P ¼ phosphorus; S.O.M ¼ soil organic matter; K ¼ potassium; N ¼ nitrogen and pH.
structed using the Simple Matching index and the UPGMA algorithm in the TREECON
program. The bootstrap values over 50% are indicated on the branches.

When comparing the T-RFLP profiles associated with plants

ecosystems is supported by various studies (Johnson, 1995; López
et al., 2003; Erickson et al., 2005, González-Ruiz et al., 2008) where
microorganisms favor the assimilation of nutrients (Davison, 1988),
produce growth-promoting hormones (Denarie et al., 1992), fix
nitrogen (Torrey, 1978; Dawson, 1986; González-Ruiz et al., 2008),
and suppress pathogens (Dobbelaere et al., 2003) amongst others.
Our results showed that soils associated with plants presented
greater nitrogen, potassium and organic matter contents than soils
without plant cover. Similarly, soils associated with actinorhizal
plants tend to present greater nitrogen content than soils associ-
ated with non-actinorhizal plants. Similar results were reported in
Sierra Nevada, California by Erickson et al. (2005), who found
a greater nitrogen content in soils associated with the actinorhizal
plant Ceanothus, than in patches of non-actinorhizal plants such as:
Abies concolor, Calocedrus decurrens, Pinus lambertian, C. cordulatus
and A. patula. However, the influence of plants over the soil
communities does not necessarily result in an increase in the
microbial diversity since soils without plant cover are an important
reservoir of bacterial richness within the ecosystem. These results
coincide with those reported in the literature wherein it has been
observed that soil without plant cover showed a similar diversity
(Kuske et al., 2002) or slightly greater diversity (Duineveld et al.,
2001; Kowalchuk et al., 2002) than those associated with plants,
thereby maintaining the idea that the presence of plants is a
selection factor for determined phylotypes.
(samples A and NA) in both sampling times, it was found that the
number of T-RFs in October were lower than in the month of May.
This result is in agreement with the tendency toward higher organic
matter content that was observed in fall. An explanation to the
difference between T-RFLP profiles could be a result of changes in the
substrate availability leading to a greater competition, where phy-
lotypes that are better adapted to the environmental conditions,
represented by these T-RFs, become dominant in the community
and therefore, reduce the existing diversity. Some studies suggested
that the competitive interaction of the carbon source is an influential
factor in the diversity of species in the microorganism communities,
and that it could favor the dominance of the better adapted groups
(Zhou et al., 2002; Treves et al., 2003). In the fall, senescent plants
provide a pulse of labile carbon to support microbial growth and
variation in the chemistry of these compounds provides a potential
carbon source to promote diversity in the microbial community
(Bowman and Steltzer, 1998). In spring, on the other hand, the rapid
changes in microclimate and the exhaustion of labile C compounds
could lead to a turnover of the microbial community (Lipson et al.,
1999). In fact, seasonal changes in the microbial communities
associated with changes in the development of the plant cover have
been described in different ecosystems (Jaeger et al., 1999; Schadt
et al., 2003; Bowman et al., 2004).
Our data also showed that the bacterial richness in soil without
plant cover showed significant seasonal differences, with a greater
1122 F. Farı́as et al. / Journal of Arid Environments 73 (2009) 1117–1124

a plant tissues, therefore it has been suggested that plants actively

70 recycle and transport K to the superficial horizon of the soil (Job-
bágy and Jackson, 2001).
60
We also analyzed the T-RFLP profiles using the MiCA tools to
50 detect the possible phylotypes in order to infer the community
40 composition. Although, the association of a T-RF to a determined
phylotype is not foolproof since two different phylotypes may have
30 the same restriction site in the 16S rRNA gene, or one phylotype can
20 have more than one copy of the 16S rRNA gene and therefore
produce more than one peak, we considered the characterization of
10
the bacterial composition in terms of the main bacterial lineage.
0 Our data showed that in the soil samples of the matorral, Firmicutes
Alphaproteobacteria

Betaproteobacteria

Deltaproteobacteria

Gammaproteobacteria

Firmicutes

Actinobacteria

Bacteroidetes

Acidobacteria

Cyanobacteria

ND

Others
and Proteobacteria were the dominant classes. Traditionally, using
culture-dependent methods, the Firmicutes were considered the
most abundant group in all soil types (Janssen, 2006). However, the
use of culture-independent techniques has shown this group to be
less numerous suggesting that they are either difficult to cultivate
or their cells are more difficult to lyse and hence, more difficult to
detect with methods based on DNA-PCR. Thus, previous studies on
b the microbial composition of arid and semi-arid soils, have found
70 a domain of the Acidobacteria phylum. In the case of topsoils in the
60 Sonoran Desert, Nagy et al. (2005) reported 51% Acidobacteria,
15.5% Proteobacteria, 13.3% Flexibacteria and relatives, 6.7% Acti-
50
nobacteria, 4.5% Planctomycetes, and 8.9% unknown. In the Mojave
40 Desert (California), the Acidobacteria represented almost 20% of the
30 relative abundance followed by Alphaproteobacteria (7%), Actino-
bacteria (5%), Betaproteobacteria (4%) and Firmicutes (4%) (Fierer
20
et al., 2005). Also, in Arizona soils, Acidobacteria represented 50% of
10 the clones obtained, a superior value when compared to Proteo-
bacteria (Dunbar et al., 1999, 2002). These results suggest that
0
the sclerophyllous matorral of central Chile could represent an
Alphaproteobacteria

Betaproteobacteria

Deltaproteobacteria

Gammaproteobacteria

Firmicutes

Actinobacteria

Bacteroidetes

Acidobacteria

Cyanobacteria

ND

Others

ecosystem with distinctive characteristics with respect to the

structure of the main soil bacterial groups, where the presence of
vegetational patches composed of pioneer actinorhizal plants such
as C. hystrix could play a key role in the conformation of microbial
communities of the superficial horizon of the soil.
The data obtained in this work also showed that an important
Fig. 3. Percentage of main microbial classes identified using the MiCA tool for each number of the T-RFs were not identified in the database, suggesting
sample type: (a) May, (b) October. Black bars: actinorhizal (A), light grey bars: non- that these habitats could be colonized by unique semi-arid soil
actinorhizal (NA), dark grey bars: interspace (I) soil samples. bacteria. Therefore, more information on the changes in the
diversity and composition of the soil bacterial community associ-
diversity in October. This could arise from differences in rainfall ated with changes in the soil environment is necessary to under-
between both seasons. According to the 30-year registry of rainfall stand the functioning of these semi-arid ecosystems. Additionally,
taken at the weather station closest to the sampling site (‘‘La Obra sequence analysis of the non-identified T-RFs which could be
del Maipo’’), the average precipitation during May exceeds three unique to these environments will help define more precisely the
times that in October. It has been suggested that the soil matrix composition of the bacterial communities of these soils.
consists of a set of spatially isolated islands populated by microbes,
particularly during the part of the year when rainfall is scarce Acknowledgments
(Becker et al., 2006). Hence, in the superficial layers of the soil the
spatial isolation and heterogeneity of the resources at the micro- This work was supported by Fondecyt projects No 1040880/
habitat level could explain the high microbial richness observed 1080280 and ENL-DID 07/16 Project, U. Chile. The authors thank
(Zhou et al., 2002, 2004). This could be a particularly relevant J. Leal for technical assistance.
phenomenon in the interspace soil where the composition of the
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