Applied Soil Ecology 28 (2005) 191–201

www.elsevier.com/locate/apsoil

Microbial community composition and functioning in the

rhizosphere of three Banksia species in native
woodland in Western Australia
Petra Marschnera,*, Pauline F. Griersonb, Zed Rengelc
a
Soil and Land Systems, School of Earth and Environmental Sciences,
The University of Adelaide, DP 636, Adelaide, SA 5005, Australia
b
Ecosystems Research Group, School of Plant Biology, University of Western Australia,
35 Stirling Highway, Crawley, WA 6009, Australia
c
Soil Science and Plant Nutrition, School of Earth and Geographical Sciences,
University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
Accepted 4 September 2004

Abstract

Annual plant species differ in their rhizosphere microbial community composition. However, rhizosphere communities are
often investigated under controlled conditions, and it is unclear if perennial plants growing in the field also have rhizosphere
communities that are specific to a particular plant species. The aim of our study was to determine the bacterial community
composition of three species of Banksia (B. attenuata R. Brown, B. ilicifolia R. Brown and B. menziesii R. Brown) growing in
close proximity in a native woodland in Western Australia and to relate community structure to function. All three species are
small trees that produce cluster roots in the field following winter rains. Cluster roots and rhizosphere soil were sampled in early
spring (August 2001) and again four weeks later (September 2001). Many new cluster roots were formed in the period between
the August and the September sampling. Rhizosphere soil pH, percent soil moisture and C and N content did not differ
significantly among species or sampling times. However, the bacterial community composition on the cluster roots and in the
rhizosphere soil, studied by denaturing gradient gel electrophoresis (DGGE), differed among the three species, with cluster root
age class (young or mature to senescing) and also between sampling times. These changes in community composition were
accompanied by changes in the activity of some of the enzymes studied. The activities of b-glucosidase and protease increased
over time. The three species differed in asparaginase activity, but not in the activity of acid and alkaline phosphatase in the
rhizosphere. These results suggest a relationship between the changes in composition and function of bacterial communities.
# 2004 Elsevier B.V. All rights reserved.

Keywords: Bacterial community composition; Banksia; Cluster roots; DGGE; Enzyme activity; Rhizosphere

* Corresponding author. Tel.: +61 8 8303 7379; fax: +61 8 8303 6511.
E-mail address: petra.marschner@adelaide.edu.au (P. Marschner).

0929-1393/$ – see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2004.09.001
192 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201
1. Introduction
Cluster or proteoid roots are formed by a range of
plant species, including members of the Proteaceae,
Casuarinaceae, Mimosaceae, Fabaceae, Myricaceae
and Moraceae (Dinkelaker et al., 1995). Cluster roots
comprise closely spaced lateral rootlets along first
order laterals; these rootlets have limited growth and
are densely covered with root hairs (Purnell, 1960;
Lamont, 1973, 2003; Gardner et al., 1983; Dinkelaker
et al., 1995). Many species that form cluster roots are
adapted to nutrient-poor soils, especially those low in
available phosphorus and nitrogen. Cluster roots are
formed under low P supply (Groves, 1964; Beadle,
1968; Grundon, 1972; Gerke et al., 1994; Neumann et
al., 1999, 2000; Shane et al., 2003) but to some extent
also under Fe deficiency and low N (Gardner et al.,
1982; Rosenfield et al., 1991). High P supply almost
completely inhibits cluster root formation (Groves,
1964; Grundon, 1972; Groves and Keraitis, 1976;
Neumann et al., 1999, 2000).
Banksia and other members of the Proteaceae form
cluster roots predominantly in the uppermost layer of
soil, especially in the humus layer (Lamont, 1973) and
proliferate at the soil–litter interface (Jeffrey, 1967;
Lamont, 1973; Grierson and Attiwill, 1989). Banksias
are dominant in many ecosystems with low nutrient
availability in Australia, with 76 species occurring
across a range of vegetation types, including tropical
forest and dry heathlands. In natural Banksia wood-
land in Western Australia, cluster root formation is
highly seasonal, with new roots being initiated after
the winter rains (Grierson and Adams, 2000). The
formation of lateral roots appears to be stimulated by
highly localised nutrient release from decomposing
organic matter. It has also been proposed that
microorganisms may play an important role in cluster
root formation (Lamont and McComb, 1974; Malajc-
zuk and Bowen, 1974). However, cluster roots are also
formed under sterile conditions albeit to a lesser extent
than in non-sterile conditions (Gardner et al., 1982).
Plant growth regulators such as auxins also appear to
be important in the induction of cluster roots (Gilbert
et al., 1999).
In Banksia woodlands, cluster roots often form
mats with high rooting density and thorough
exploitation of the soil. The rhizosphere of cluster
roots of Banksia is characterised by high concentra-
tions of low molecular weight organic anions and
phenolics that mobilise nutrients (Grierson, 1992;
Roelofs et al., 2001) and are also easily available
carbon sources for microorganisms in the rhizosphere
(Jones and Darrah, 1994). In addition, organic
phosphates in the rhizosphere is mineralized by acid
phosphatase exuded by cluster roots at high rates
(Grierson and Adams, 2000; Taranto et al., 2000).
Many studies, mainly with annual plants growing
under controlled conditions, have shown that plant
species differ in exudate amount and composition
(Rovira, 1959; Klein et al., 1988; Grayston et al.,
1996; Merbach et al., 1999) and also in rhizosphere
community composition (Grayston et al., 1996;
Westover et al., 1997; Germida et al., 1998; Ibekwe
and Kennedy, 1998; Miethling et al., 2000; Marschner
et al., 2001a). Direct evidence that root exudate
composition can affect soil microbial community
composition was obtained recently for low molecular
weight organic anions in the rhizosphere of cluster
roots of white lupin (Marschner et al., 2002) and
following addition of artificial root exudates (Baudoin
et al., 2003).
The functional capacity of the soil microbial
community, as reflected in the activities of enzymes
involved in nutrient mineralization processes, varies
among soils dominated by different plant species
(Waldrop et al., 2000; Kourtev et al., 2003). Never-
theless, there have been relatively few studies that
have examined root exudation, microbial rhizosphere
community composition and enzyme activities of
perennial plants under field conditions (Kourtev et al.,
2003). Recently, Grierson and Adams (2000) demon-
strated seasonal shifts in acid phosphatase activity,
microbial biomass and ergosterol content (an index of
living fungal biomass) in the surface soils of jarrah
(Eucalyptus marginata) forest in Western Australia
with or without the understorey species Banksia
grandis. Their results indicate that microbial com-
munity composition and activity are strongly affected
by plant species, the season as well as the availability
of substrate and water.
It is generally accepted that the link between
microbial community composition and function is
often weak because many ecosystem functions are
carried out by a wide range of microbes, which form
substrate guilds (Zak et al., 1994; Nannipieri et al.,
2002). Consequently, changes in the abundance of a
 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201
single species often have little effect on a given
function (Nannipieri et al., 2002; Avrahami et al.,
2003; Miethling et al., 2003). However, there are
indications that changes in overall microbial commu-
nity composition can be correlated with changes in
certain functions both under controlled conditions
(Kandeler et al., 2002; Avrahami et al., 2003) and in
the field (Marschner et al., 2003).
The objective of this study was to determine
bacterial rhizosphere community composition and
enzyme activities in three Banksia species growing
close to each other in a natural Banksia woodland
north of Perth, Western Australia. Banksia is an
interesting model for understanding interactions
among rhizosphere microorganisms and plant adapta-
tions to low nutrient soils. We examined three co-
occurring species of Banksia for differences in
bacterial rhizosphere community structure in relation
to the activity of several enzymes involved in C, N and
P cycling. Similarity among species would indicate
that there is a generalized effect of Banksia on nutrient
cycling processes while differences would suggest that
each Banksia species has a distinct effect on soil
microorganisms and function in the rhizosphere.
2. Materials and methods
The sampling site was a native woodland at
Wanneroo, approximately 20 km north of Perth
(318450 S, 1158480 E). Soils are nutrient-poor siliceous
sands that dry out to great depth during summer, with
nutrients concentrated in the litter layer and the top 0–
5 cm of soil (Lamont, 1993; Pate et al., 1998). The
three Banksia species studied (B. attenuata R. Brown,
B. ilicifolia R. Brown and B. menziesii R. Brown) are
small trees of 3–7 m height with hard sclerophyllous
leaves. Cluster roots and rhizosphere soil were
sampled in mid-August 2001 (early spring) and again
4 weeks later in September 2001. The August
sampling was at the beginning of the seasonal growth
period (Grierson and Adams, 2000), with only a few
new cluster roots initiated; the majority of cluster roots
were from the previous year (dark-colored cluster
roots and no longer active). At the second sampling in
September, numerous new cluster roots had been
formed at the soil–litter interface by all three Banksia
species. Samples of roots and soils in the top soil layer
193
(0–5 cm) were taken from beneath the Banksias at four
different locations within an area of approximately
1 ha. At each location, the three species grew within
2–3 m distance from each other. Sampling locations
differed between the two sampling dates. In order
to ensure that the sampled roots belonged to the
correct species, the samples were taken within 50 cm
of the stem of each species and the lateral root
traced back to the parent tree. Root mats with adhering
soil were carefully extracted, put in plastic bags,
placed on ice and processed within 3 h of collection.
Size, color and location along the cluster roots were
used to differentiate between young (small, light
brown, close to root tip of the laterals) and mature
cluster roots (larger, dark brown, further away from
the root tip).
Lightly adhering soil was removed by shaking the
roots gently and then two young cluster roots or one
mature cluster root were placed in microcentrifuge
tubes and freeze-dried for determination of the
bacterial community composition. The lightly adher-
ing soil from old and young cluster roots was mixed
(hereafter referred to as rhizosphere soil) and sieved to
2 mm. Approximately 500 mg of rhizosphere soil
were placed in microcentrifuge tubes and freeze-dried
for determination of the bacterial community compo-
sition. The rhizosphere soil for the chemical analyses
and enzyme activity measurements was stored at 4 8C
until further analysis. Bulking of the rhizosphere soil
from both cluster root ages was necessary because the
amount of soil from each cluster root age would have
been insufficient for analyses of all properties
examined in this study.
At the second sampling only, soil was also
collected from four open areas between trees adjacent
to the locations where the Banksia cluster roots were
sampled. This soil was free of vegetation and litter and
is referred to hereafter as bare soil. Samples of bare
soil contained some fine roots, but no cluster roots.
2.1. Chemical properties of the rhizosphere
and bare soil
Soil moisture content was measured after drying
at 105 8C for 5 h and given on a gravimetric basis.
Soil pH was determined in a water:soil ratio of 1:1.
Total C and N were determined using a Carlo Erba
NA 1500 Elemental Analyzer (Milan, Italy).
194 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201
To determine water-soluble C, 20 g oven dry
weight equivalents of fresh soil samples were shaken
with 80 ml of ultrapure water for 1 h and then
centrifuged for 15 min at 3000 rpm. The supernatant
was first filtered through a Whatman #42 filter to
remove larger particles followed by filtration through
a 0.45 mm-pore membrane (Millipore, USA). Total
carbon in the extract was measured using a TOC
analyzer (Shimadzu, 5000a).
2.2. Enzyme activities in the rhizosphere and
bare soil
Protease activity was measured according to Ladd
and Butler (1972). Briefly, 5 mL Tris buffer (pH 8.1)
and 5 mL caseinate solution (2% (w/v)) were added to
1 g of moist soil. The samples were shaken for 2 h
at 50 8C. After incubation, 5 mL trichloric acid (15%
(v/v)) were added and centrifuged for 10 min at
12,000 rpm. After alkalinization of the supernatant,
tryrosine was determined photometrically at 700 nm
using the Folin-Ciocalteu reagent (33%).
L-Asparaginase activity was measured according
to Frankenberger and Tabatabai (1991). Nine
milliliters of Tris buffer (pH 10) and 1 mL of L-
asparagine solution (0.5 M) were added to 5 g of
moist soil. Samples were incubated at 37 8C for 2 h.
After incubation, 40 mL KCl (2.5 M)–Ag2SO4
(100 mg L1) solution were added. Ammonium con-
centration was determined by steam distillation.
The method by Alef and Nannipieri (1995) was
used for measurement of b-glucosidase activity. One g
moist, sieved soil was incubated with 4 mL modified
universal buffer (pH 6.0) and 1 mL p-nitrophenyl-b-D-
glucoside (25 mM) solution. After incubation at 37 8C
for 1 h, 1 mL CaCl2 solution (0.5 M) and 4 mL Tris
buffer (0.1 M, pH 12) were added. The nitrophenol
concentration was determined photometrically at
400 nm.
Phosphatase activities were determined according
to Tabatabai and Bremner (1969). Alkaline phospha-
tase activity was measured at the second sampling in
September only. One g of moist fresh soil was
incubated with 4 mL modified universal buffer (pH 6.5
for acid phosphatase, pH 11 for alkaline phosphatase)
and 1 mL p-nitrophenyl phosphate (15 mM) for 1 h at
37 8C. After incubation, 1 mL CaCl2 (0.5 M) and
4 mL NaOH (0.5 M) were added to stop the reaction
and to increase the pH. The nitrophenol concentration
was determined photometrically at 400 nm.
Enzyme activities were compared by one-way
ANOVA for the two sampling dates separately and by
two-way ANOVA for both sampling dates together
using the least significant difference to compare the
mean values (GenStat 5th edition, Rothamsted
Experimental Station, 2000).
2.3. Assessment of the bacterial community
composition in the rhizosphere soil and on
cluster roots
The method described in Marschner et al. (2001b)
was used for bacterial community composition
analysis by polymerase chain reaction-denaturing
gradient gel electrophoresis (PCR-DGGE). Briefly,
200 mM phosphate buffer and 10% (w/v) SDS were
added to the freeze-dried root or rhizosphere soil
samples followed by homogenization in a bead beater
(Fast-Prep, Model FP120, Qbiogene) at 5.5 m s1 for
30 s. After proteins were removed with a protein
precipitation solution (PPS1, Qbiogene), the DNA
was bound to a silica matrix (Binding matrix1,
Qbiogene), washed twice with an ethanol–salt solution
(SEWS1, Qbiogene) and then desorbed into sterile
water. The DNA samples were stored at –20 8C for
further analysis. The DNA extract was diluted 10-fold
to reduce the concentration of compounds that may
inhibit the amplification.
A highly variable section of the 16S rDNA was
amplified by using the primer set F984 and R1378
(Heuer et al., 1997). A GC clamp (Sheffield et al.,
1989) was attached to primer F984 to prevent
complete separation of the strands in the DGGE
gel. For some samples, namely young cluster roots
sampled in September, amplification of root DNA
extracts was not successful, probably due to high
concentrations of inhibiting substances such as
phenolics. For samples that could be amplified, the
resulting fragments were separated by DGGE. To
obtain an adequate number of replicates, in some cases
DNA extracts from more than one cluster root of a
Banksia species at a given location was amplified. The
community-specific band patterns were digitized,
normalized and analyzed by multivariate analysis
(for details see Marschner et al., 2001b) (CANOCO
4.0, Microcomputer Power, Ithaca, USA). We used
 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201 195

principal component analyses (PCA) to assess the
general differences between band patterns. Redun-
dancy discriminate analyses (RDA) provides more
information because not only banding patterns of
different samples are compared but RDA also takes
environmental factors such as plant species, time, soil
moisture and activity of enzymes related to this
sample into account. The Monte Carlo Permutation
test calculates the proportion of variation explained
and the significance of a given environmental factor,
and thus its importance to community composition.
Even though these are only mathematical correlations,
they can, in most cases, be interpreted in a biological
meaningful way (Jongman et al., 1995).
3. Results
In August 2001 (the first sampling), only few young
cluster roots were found, with the majority being old,
dark-colored cluster roots. In the 4 weeks between the
first and the second sampling, many new cluster roots
were formed by all three Banksia species directly at
the soil–litter interface.
Moisture content of the rhizosphere soil of all three
species ranged between 6.2 and 7.5% (w/w) and was
similar between sampling times. The moisture content
of the bare, vegetation-free soil was only determined
for the September sampling and was significantly
lower (2.8% (w/w)). The pH of the rhizosphere soil at
the second harvest ranged between pH 4.6 and pH 5.1
and did not differ significantly among species, or
between rhizosphere and the bare soil. Water-soluble
C content in the rhizosphere soil ranged between 9
and 13 mg L1 and did not differ among species or
sampling time. Total C and N contents of the
rhizosphere soil were similar among the three species
and between sampling times, and ranged between 10
and 17 g kg1 C and 0.4 and 0.7 g kg1 N, giving a
C/N ratio of 22–28.
After initiation of new cluster roots in September,
b-glucosidase and protease activities were signifi-
cantly greater than in August, whereas the activities of
acid phosphatase and asparaginase activity did not
change over time. When both sampling dates were
analyzed together, the three species significantly
differed in asparaginase activity. At the August
sampling, there was no significant difference in
enzyme activities of the rhizosphere soils among
the three Banksia species (Table 1). In September,
asparaginase activity differed significantly between
the species, being more than three times greater in
B. menziesii than in B. attenuata. Compared to the
rhizosphere soil, acid phosphatase, b-glucosidase and
protease activities were about three times lower in the
bare soil.
Thirty different bands could be distinguished in the
DGGE gels, with each sample containing between 5 and
14 bands. When the DGGE banding patterns of the
bacterial communities from both sampling dates were
analyzed together, all environmental variables used in
the redundancy discriminate analysis (RDA) had a
significant effect on the bacterial community composi-
tion. Plant species (explaining 8% of the total variation
within the banding patterns), location (root or rhizo-
sphere soil) (5%) and root age (6%) had the strongest

Table 1
Activities of asparaginase b-glucosidase, acid and alkaline phosphatase and protease in the rhizosphere soil of Banksia attenuata, B. ilicifolia
and B. menziesii in August and September (means of four replicates  standard error)
Asparaginase b-Glucosidase Acid phosphatase Alkaline phosphatasea Protease
(mg N g1 soil) (mg nitrophenol g1 soil) (mg nitrophenol g1 soil) (mg nitrophenol g1 soil) (mg tyrosine g1 soil)
August
B. attenuata 81 166  24 3677  448 – 732  262
B. ilicifolia 71 250  56 3669  828 – 627  163
B. menziesii 62 163  24 2994  475 – 619  139
September
B. attenuata 3  1 323  82 3774  1107 506  112 1044  225
B. ilicifolia 6  2 342  60 5089  1267 577  152 1401  356
B. menziesii 10  1 370  143 4258  1447 426  83 1225  564
Bare soil 5  2 96  4 1429  128 411  65 213  65
a
Alkaline phosphatase activity was only determined in September.
196 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201

Table 2
Results of the Monte Carlo Permutation test in percent of variation explained and significance of environmental factors (P value) for the bacterial
community composition on the roots of B. attenuata, B. ilicifolia and B. menziesii in August and September determined by denaturing gradient
gel electrophoresis (DGGE)
Both dates August September Average (%)
% P % P % P
** ** **
Banksia species 8 11 14 11
** a a
Sampling time 3 3
** * **
Rhizosphere soil vs. root 5 5 12 7
** ** a
Root age 6 6 6
** ** **
Soil moisture 4 11 11 9
** a **
pH 3 17 10
** ** **
Asparaginase 3 11 12 9
** ** **
Protease 3 10 10 8
** a **
Alkaline phosphatase 3 16 6
** ** **
Acid phosphatase 4 11 10 8
** ** **
Glucosidase 4 8 11 8
Data sets of the two sampling dates were compared for the two dates together or separately. The average percent of variation explained represents
the average of the comparisons; ns indicates non-significant at P  0.10.
*
P  0.05.
**
P  0.01.
a
Correlation could not be calculated.

effect (Table 2). The community composition on the
cluster roots and in the rhizosphere soil also changed
with time in all three species. The bacterial community
composition was correlated with enzyme activities,
especially with b-glucosidase and acid phosphatase
activity (explaining 4% of the total variation). The RDA
plot shows that the community composition of B.
attenuata differed clearly from that of B. ilicifolia and B.
menziesii; in all three species the communities changed
from August to September (Fig. 1). The bacterial

Fig. 1. Ordination plot of bacterial communities in the rhizosphere soil and the cluster roots (young or mature) of B. attenuata, B. ilicifolia and B.
menziesii in August and September generated by redundancy discriminate analysis (RDA) of 16S rDNA denaturing gradient gel electrophoresis
(DGGE) profiles. The values on the axes refer to the % of the total variance explained by the axis. Communities are represented as symbols.
Communities (symbols) close to each other have similar compositions, whereas communities far apart differ strongly in composition.
 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201
communities of the young cluster roots of B. ilicifolia
and B. menziesii differed from those of the old cluster
roots in the August sampling.
Although many young cluster roots were found in
September, none of their DNA extracts could be
amplified. Amplification of DNA extracts of mature
cluster roots of B. attenuata and B. ilicifolia from the
September sampling were also not successful; hence,
only the rhizosphere soil samples of these two species
were included in the analysis.
In order to obtain a clearer picture of the relation-
ship between bacterial community composition and
environment, the two sampling dates were analyzed
separately. In August, the bacterial community
composition was affected by Banksia species and
soil moisture (each explaining 11% of the total
variation of the banding patterns) and to a lesser extent
by root age (6%) and location (5%) (Table 2). The
bacterial community composition was correlated with
the activities of all enzymes. The PCA plot shows that,
in August, the community composition of B. ilicifolia
differed from that of B. menziesii whereas that of
B. attentuata showed similarities with B. ilicifolia and
B. menziesii (Fig. 2). In B. ilicifolia, the communities
of the young cluster roots and those in the rhizosphere
soil differed from those of the old cluster roots. In
B. menziesii the community of the young cluster
root differed from those of the old roots and the
rhizosphere soil. However, it should be noted that
Fig. 2. Ordination plots of bacterial communities in the rhizosphere soil and the cluster roots (young or mature) of B. attenuata, B. ilicifolia and
B. menziesii in August (A) and September (B) separately generated by principal component analysis (PCA) of 16S rDNA denaturing gradient gel
electrophoresis (DGGE) profiles. For explanation see Fig. 1.
197
only one DNA extract of young cluster roots from
B. menziesii could be amplified. Amplification of
the DNA extracts of the young cluster roots of
B. attenuata was not successful, but the bacterial
communities of the old cluster roots and those of the
rhizosphere soil differed from each other.
In September, the bacterial community composi-
tion was affected by pH (explaining 17% of the total
variation), Banksia species (14%), location (12%) and
soil moisture (11%) (Table 2). Community composi-
tion was correlated with the activity of all enzymes
measured, most strongly with that of alkaline
phosphatase (explaining 16% of the total variation).
The effect of root age could not be examined, because
DNA extracts from young cluster roots sampled in
September could not be amplified. The PCA plot of
the September sampling shows that the bacterial
communities of B. menziesii differ from those of
B. ilicifolia and B. attenuata, and the bare soil com-
munities were different from those in the rhizosphere
soil (Fig. 2).
To assess the overall importance of the measured
environmental factors, the mean percent explanation
was calculated (Table 2). Bacterial community
composition was most strongly affected by Banksia
species (explaining 11% of the total variation), soil pH
(10%) and moisture (9%) and was correlated with the
activity of all enzymes measured to a similar extent
(6–9%).
198 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201
4. Discussion
The rhizosphere soil of Banksia species growing
close to each other differ in rhizosphere bacterial
community composition and asparaginase activity
(Fig. 1, Table 1). The bacterial community composi-
tion changed over time, which was reflected in
changes in b-glucosidase and protease activity. The
strong correlation between bacterial community
composition and enzyme activities also suggests that
bacterial community composition and function are
linked (Table 2).
Plant species-specific microbial rhizosphere com-
munities have been previously described for studies
where different species were grown separately (West-
over et al., 1997; Germida et al., 1998; Grayston et al.,
1996; Ibekwe and Kennedy, 1998; Miethling et al.,
2000; Marschner et al., 2001a). Differences among
plant species may be attributed to differential amount
and composition of root exudates (Marschner et al.,
2002; Baudoin et al., 2003). Roelofs et al. (2001)
compared the exudation of cluster roots of Banksia
occidentalis and B. prionoites under controlled
conditions. Even though the total carboxylate exuda-
tion from the roots did not differ, cluster roots of
B. occidentalis exuded relatively more cis- and trans-
acconitate and those of B. prionoites exuded more
malonate, lactate and acetate. Our findings that the
Banksia spp. differ in rhizosphere community
composition indicate that there may also be differ-
ences in root exudation on between species under field
conditions. However it should be noted that under field
conditions variables such as soil type and texture,
micro-site variation in nutrient availability and soil
moisture will also influence rates of root exudation and
their chemical composition.
The bacterial community composition differed
between young and mature cluster roots (Fig. 1).
Similar changes in microbial community composition
with root age have been shown in cluster roots of white
lupin (Marschner et al., 2002) and roots of canola,
chickpea and Sudan grass (Marschner et al., 2001a;
Rumberger and Marschner, 2003) and can be related
to changes in root exudate composition with root
age (Neumann et al., 1999, 2000; Rumberger and
Marschner, 2003). In the present study the old cluster
roots may have been non-functional and already
partially decaying which could have resulted in
changes in microbial community composition due
to occurrence of saprophytic microorganisms.
In this study we observed changes in the
composition of the bacterial rhizosphere community
over time (Figs. 1 and 2). This is in accordance with
other studies with maize and white lupin where the
microbial community composition changed with
plant age (Marschner et al., 2001b, 2002). Temporal
changes in rhizosphere microbial community compo-
sition are likely to be at least partially attributable to
changes in exudate amount and composition, both of
which change with plant age and/or plant develop-
mental stage (Aulakh et al., 2001). In the present
study, many new active cluster roots were formed
during 4 weeks between the August and September
sampling. It may be assumed that these young roots
differed from the old roots in both root exudation and
nutrient uptake, as it has been shown in other studies.
Exudation rate in cluster roots of white lupin is highest
in mature cluster roots (Neumann et al., 1999) and
Häussling et al. (1990) showed that NO3 and P uptake
are highest at the root tip of Norway spruce. Thus, in
the present study conditions for the microorganisms
colonizing the rhizosphere would have changed
dramatically within these 4 weeks. Besides root
exudation, other important factors influencing micro-
bial communities include, for example, soil moisture
(Table 2) (Rumberger and Marschner, 2003), soil pH
(Table 2, Marschner et al., 2004), nutrient availability
(Liljeroth et al., 1990) and predation (Griffiths et al.,
1999).
b-Glucosidase and protease activities in the
rhizosphere soil increased from the first to the second
sampling (Table 1), which could be due to changes in
root exudation and thus microbial activity. Addition-
ally, the observed changes in bacterial community
composition may have contributed to this increase (see
below). For example, acid phosphatase activity in soils
from jarrah (E. marginata) forest in Western Australia
changes seasonally, primarily in response to soil water
content and its consequent effects on root growth and
microbial activity (Grierson and Adams, 2000).
Seasonal changes in acid phosphatase activity also
differed among plant species and were correlated with
shifts in fungal versus bacterial dominance within the
microbial community (Grierson and Adams, 2000).
In the present study, activities of acid phosphatase,
protease and b-glucosidase were higher in the
 P. Marschner et al. / Applied Soil Ecology 28 (2005) 191–201
rhizosphere soil than in the vegetation-free soil. This
stimulation of microbial activity in the rhizosphere is
in agreement with earlier studies (Tarafdar and Jungk,
1987; Kandeler et al., 2002).
Changes in bacterial community composition were
related to differences in the activity of some of the
enzymes studied. The differences in bacterial com-
munity composition among the Banksia species were
reflected in significant differences in asparaginase
activity. The change in community composition over
time was associated with an increase in activities of b-
glucosidase and protease from August to September.
At the September sampling, the vegetation-free bare
soil had lower acid phosphatase, glucosidase and
protease activity than the rhizosphere soil (Table 1)
and differed in bacterial community composition
from the rhizosphere soil (Fig. 2). A link between
community composition and function is also indicated
by the strong correlation between bacterial commu-
nity composition and enzyme activity (Table 2).
Recently, Kandeler et al. (2002) found that decreasing
activities of acid and alkaline phosphatase and
invertase with increasing distance from the root
surface were associated with changes in bacterial
community composition.
However not all enzyme activities showed con-
sistent links with bacterial community composition in
the present study. For example, acid phosphatase
activity did not change over time and did not differ
between Banksia species. This may be due to fact that
acid phosphatase is also released by plant roots and
fungi and the activities measured here cannot be solely
attributed to the bacterial community (Grierson and
Adams, 2000). Alternatively, decreases in abundance
and/or activity of a certain species can be compensated
for by increases in abundance and/or activity of
another species with a similar function, i.e. there is
functional redundancy (Zak et al., 1994; Nannipieri et
al., 2002). Miethling et al. (2003) also demonstrated
that microbial community composition and function
(substrate utilisation pattern) are not necessarily
correlated. Changes in metabolic processes may occur
without concomitant changes in microbial community
structure. For example, nitrification rates may increase
without short-term changes (days) in the community
structure of ammonia-oxidizing bacteria, indicating
that only the activity of the species already present
increased (Avrahami et al., 2003). However, over
199
longer periods (weeks), both nitrification rates and the
community structure of ammonia-oxidizing bacteria
changed (Avrahami et al., 2003). It should be noted
that due to the high temporal and spatial heterogeneity
of ecosystems (Ettema and Wardle, 2002), the concept
of functional redundancy may only hold on a larger
scale (cm to m), whereas in a particular niche (for
example in the rhizosphere) the presence of a certain
species may be crucial for a certain function.
In conclusion, this study demonstrated that the
rhizospheres of Banksia species growing under field
conditions in Western Australia differed in bacterial
community composition and that the bacterial com-
munity changed rapidly with the onset of new root
growth after winter rains. Shifts in bacterial commu-
nity composition were associated with changes in
activity of enzymes involved in N and C cycling. Thus
microbial community composition and enzyme
activity are highly variable in time and space. In
the rhizosphere, the main driving factors for changes
in bacterial community composition and activity are
likely to be root exudate amount and composition.
Consequently, each plant species may modify the
conditions in the rhizosphere in order to maximize
nutrient acquisition from organic matter by promoting
particular functional groups of microorganisms.
Acknowledgements
This study was supported by the Australian
Research Council (IREX X00001664). We thank
Dr. Nui Milton (University of Western Australia) for
the analysis of total C and N and water-soluble C.
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