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Bacillus spp. and Pisolithus tinctorius effects on Quercus ilex
ssp. ballota: a study on tree growth, rhizosphere community
structure and mycorrhizal infection
Jezabel Domenech, Beatriz Ramos-Solano, Agustin Probanza,
Jose
´Antonio Lucas-Garcı
´a, Juan Jose
´Colo
´n, Francisco Javier Gutie
´rrez-Man
˜ero
*
Departamento CC. Ambientales y RR. Naturales, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU,
Urbanizacio
´n Monteprı
´ncipe, Ctra. Boadilla Km 5.3, Boadilla del Monte, P.O. Box 67, Madrid 28668, Spain
Received 5 February 2003; received in revised form 14 October 2003; accepted 23 February 2004
Abstract
A local species of oak (Quercus ilex ssp. ballota) was inoculated or co-inoculated with the ectomycorrhizal fungus Pisolithus
tinctorius and two plant growth promoting rhizobacteria (PGPR) of the genus Bacillus (Bacillus licheniformis CECT 5106 and
Bacillus pumilus CECT 5105). Effects of inoculation on growth, on N acquisition by the plant roots, changes in rhizosphere
microbial communities and the degree of mycorrhization were evaluated. Only B. licheniformis promoted the growth of Q. ilex
seedlings while co-inoculation of either bacterial strain with P. tinctorius had a negative effect on plant growth. Furthermore, B.
licheniformis inhibited fungal growth as revealed by ergosterol/chitin analysis. As shown by phospholipid fatty acid profiles, the
inoculation caused a slight alteration in the microbial community structure of the rhizosphere, both in the total community and
the culturable populations.
#2004 Elsevier B.V. All rights reserved.
Keywords: PGPR; Mycorrhiza; Quercus ilex ssp. Ballota; Microbial community structure
1. Introduction
For many years, research on beneficial microorgan-
isms associated to plant roots has focused on mycor-
rhizal fungi (Harley and Smith, 1983; Perry et al.,
1989; Marx and Cordell, 1989; Kropp and Langlois,
1990) and symbiotic bacteria belonging to the genera
Frankia (Schwintzer and Tjepkema, 1990) and Rhi-
zobium (Torrey, 1992). Today, there is an increasing
interest in the use of beneficial bacteria as inoculants
for nursery tree seedlings (Chanway, 1997). This is
due to the repeated demonstrations of agricultural and
horticultural plant growth stimulation by plant growth
promoting rhizobacteria (PGPR) and the fact that
bacterial inoculation on forest seedlings before out-
planting is inexpensive, environmentally benign, and
easily applied in nursery treatments (Vonderwell and
Enebak, 2000). Quercus ilex ssp. ballota is the domi-
nant tree species in the mature stage of the Mediter-
ranean forest. Despite its beneficial effect on soil
through the release of easily mineralizable organic
matter, it is seldom used on reforestation programs due
to its slow growth rate and the high percentage of
seedling decay after transplanting.
Forest Ecology and Management 194 (2004) 293–303
*
Corresponding author. Tel.: þ34-913-724775;
fax: þ34-913-510496.
E-mail address: jgutierrez.fcex@ceu.es (F.J. Gutie
´rrez-Man
˜ero).
0378-1127/$ – see front matter #2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.foreco.2004.02.026
The mechanisms by which PGPR promote plant
growth are not fully understood, but are thought to
include: (a) the ability to produce plant hormones,
such as auxins (Mordukhova et al., 1991; Gutie
´rrez-
Man
˜ero et al., 1996), gibberellins (Gutierrez-Man
˜ero
et al., 2001), cytokinins (Tien et al., 1979), and
ethylene (Glick et al., 1995); (b) asymbiotic N
2
fixa-
tion (Boddey and Do
¨bereiner, 1995; Kennedy et al.,
1997); (c) antagonism against phytopathogenic micro-
organisms by production of siderophores (Scher and
Baker, 1982), b-1,3-glucanase (Fridlender et al.,
1993), chitinases (Renwick et al., 1991), antibiotics
(Shanahan et al., 1992), and cyanide (Flaishman et al.,
1996); and (d) solubilization of mineral phosphates
and other nutrients (De Freitas et al., 1997).
The effects of these bacteria on plant development
are especially interesting when co-inoculations of
mycorrhizal fungi and PGPRs are considered on
species that are obligatory mycorrhizal. Generally,
bacteria are assumed as mycorrhization helper bac-
teria (MHB) when they are unable to stimulate plant
growth in the absence of an appropriate mycorrhizal
fungi (Duponnois and Garbaye, 1991; Duponnois
et al., 1993). Bacteria clearly enhance seedling
growth by increasing the number of mycorrhizal
root tips (Garbaye, 1994). On the other hand, PGPR
strains may not affect the establishment of my-
corrhizal symbiosis, but may exhibit synergistic
effects on plant growth (Shishido et al., 1995;
Shishido and Chanway, 1998). Such effects of PGPR,
individually or co-inoculated with ectomycorrhiza,
have been evaluated in Pinus spp. (Probanza et al.,
2001).
Pisolithus tinctorius has been shown to be mycor-
rhizal with Pinus,Eucalyptus (Chan and Griffiths,
1988, 1991), Quercus,Castanopsis and Lithocarpus
(Tam and Griffiths, 1993). While in studies with oak
seedlings, mycorrhizal stimulation of growth and
uptake of mineral nutrients has been reported, the
effect of PGPRs alone or co-inoculated with ectomy-
corrhiza has not been examined.
Our working hypothesis was two-fold: (i) to eval-
uate whether inoculation or co-inoculation of P. tinc-
torius and two PGPRs of the genus Bacillus (Bacillus
licheniformis CECT 5106 and Bacillus pumilus CECT
5105) would enhance Q. ilex shoot seedling growth;
and (ii) whether inoculation would affect mycorrhizal
fungi and rhizosphere microbial communities in order
to link effect on plant growth with alterations in the
latter following inoculation.
2. Material and methods
2.1. Inoculants
Two PGPR Bacillus strains (B. licheniformis CECT
5106 and B. pumilus CECT 5105) and mycorrhizal
fungi P. tinctorius were used. The Bacillus strains
were isolated, identified (Probanza et al., 1996) and
characterized as PGPR able to produce indole acetic
acid-like compounds (Gutie
´rrez-Man
˜ero et al., 1996)
and gibberellins (Gutierrez-Man
˜ero et al., 2001).
The P. tinctorius (Pers.) Coker and Couch [Syn ¼
P. arhizus (Scop.:Pers.) Rauschert] inocula used was
MycorPlant
1
(Madrid, Spain). This is a commercial
inoculum whose composition per 100 g is: 10
8
fungal
spores, 45 g of acrilamide, 10 g of silica sand and 45 g
of humic acids (leonardite humates).
2.2. Seed and plant substrates
Q. ilex seeds were collected from ‘‘La Berzosa’’,a
mature Mediterranean forest located in Madrid
(Spain) (coordinates UTM 408380N, 38370W), during
the winter of 1998. Until use, seeds were stratified at
48C and surface sterilized by floating in 2.5% NaOCl
for 5 min, followed by five rinses with sterile distilled
water before sowing.
A peat:sand mixture 1:1 (w/w) was the potting
media used. The peat composition was: 90% black
peat, 8% plant compost, clays and sand, pH 6.0,
200 mg N/l, 200 mg P
2
O
5
/l and 150 K
2
O mg/l. Before
use, the substrate was autoclaved three times at 120 8C
for 20 min each.
2.3. Plant growth and inoculation conditions
In early spring, seeds were sown in plastic trays
filled with autoclaved vermiculite (termite no. 3) and
watered with sterile tap water. Conditions during
germination were 12 h light at 22 8C. One month after
germination, seedlings were transferred to 6 cm
6:5cm23 cm plastic pots (forest containers, Full-
Pot
1
, Mollerusa, Spain) with a 2 mm diameter mesh
at the base, filled with the sterile peat:sand mixture.
294 J. Domenech et al. / Forest Ecology and Management 194 (2004) 293–303
Throughout the experiment, plants were kept under
the same greenhouse conditions (light/dark 16/8 h,
temperature 18/15 8C), and watered twice a week with
tap water. Two hundred and 10 plants were trans-
planted to 6 cm 6:5cm23 cm plastic pots and
distributed into seven lots, one for each treatment.
The following treatments were used: B. pumilus (B1),
B. licheniformis (B2), B. pumilus and B. licheniformis
(B1B2), P. tinctorius (Pt), P. tinctorius and B. pumilus
(PtB1), P. tinctorius and B. licheniformis (PtB2), and
the non-inoculated control (C). Inoculation was carried
out when plants were 3 weeks old. Bacteria were stored
on 0.2% TSA at 4 8C. Twenty-four hours before inocu-
lation, bacteria were transferred to a liquid medium
(Nutrient Broth, Pronadisa, Spain). The culture was
centrifuged and washed with sterile Nutrient Broth,
and pellets were resuspended in sterile NaCl 0.9%
solution to obtain 10
8
CFU/g soil. For treatments with
P. tinctorius, a commercial powder inoculum (1.92 g)
was suspended in bacterial suspensions (100 ml) pre-
pared in the same way as previously described (PGPR–
fungi co-inoculation), or in sterile 0.9% NaCl (fungal
inoculation) to obtain a concentration of 6 105
spores/g soil. The bacterial, fungi, or bacterial–fungi
suspensions, in a final volume of 10 ml per plant, were
spread homogeneously on the soil surface. Control
plants were watered with sterile 0.9% NaCl solution.
2.4. Plant harvest and shoot growth analysis
Plants were harvested 3, 60, 90 and 270 days after
inoculation (S1, S2, S3 and S4, respectively). At each
sampling time, six plants per treatment were randomly
selected. The analyses performed at each sampling
time were as follows:
(a) Ergosterol and chitin analyses:Theywere
performed on treatments involving Pisolithus
(Pt, PtB1, PtB2) and in non-inoculated controls
(C). In these analyses, S1, S2 and S3 correspond
to the vegetative growth period, and results
appear as the average among the three, while
S4 refers to the winter period.
(b) Phospholipid fatty acids (PLFA)analyses: These
analyses were performed at sampling times S1,
S2 and S3.
(c) Nitrogen acquisition: Total nitrogen content was
determined only at S4.
(d) Biometric parameters: Growth parameters were
determined only at S4. Shoot length, shoot
surface area and shoot dry weight were evaluated.
2.5. Chitin and ergosterol analyses
Root systems were split from shoots and frozen at
70 8C for further analysis. Roots (1 g) were crushed
with a mortar in liquid N
2
to obtain a fine powder. The
powder was suspended in 3.0 ml of methanol and
centrifuged at 4500 rpm for 20 min at 4 8C. This was
repeated three times. Ergosterol was determined in
supernatants, whereas chitin was determined in pellets.
Chitin was measured as described by Ekbald et al.
(1998), with the following modifications. Each washed
and freeze-dried pellet was treated with 0.2N NaOH to
remove proteins and amino acids, which could inter-
fere with glucosamine determination. An acid hydro-
lysis (6N HCl, v/v) was performed at 80 8C for 6 h in
order to release glucosamine residues, followed by
neutralization with 3 M sodium acetate. Glucosamine
residues were evaluated by colorimetry at 653 nm.
Ergosterol was measured according to Salmanovicz
and Nylund (1988) and Nylund and Wallander (1992),
with the following modifications. Free ergosterol, as
well as that bound forming sterol esters in the super-
natant were measured together. The sample processing
consisted in evaporation of the methanolic fraction,
saponification (KOH 4% in ethanol, 80 8C for 30 min)
and a final partition with cyclohexane (4 ml). The
alkaline ethanolysis was stopped with 2 ml of a mix-
ture of Na
2
HPO
4
and KH
2
PO
4
(0.1 g/ml). The organic
phase was evaporated under a stream of N
2
and the
final dried residue was stored at 20 8C and dissolved
in 200 ml of methanol prior to analysis. Ergosterol was
separated in a two-pump Beckman HPLC provided
with a diode array detector, in a C18 reversed phase
column (150 mm 4:5 mm, i.d. in mm), and detected
at 282 nm. The mobile phase was 100% methanol
(HPLC-grade) with a gradient flow rate, which began
with 1.5 ml/min for 3 min, to decrease to 1.0 ml/min
for 5 min. The chromatographic run lasted 12 min;
ergosterol retention time was 8.5 min.
2.6. Phospholipid fatty acid analysis
For each sampling time and treatment, 1 g of rhizo-
sphere soil was obtained by gently washing the roots
J. Domenech et al. / Forest Ecology and Management 194 (2004) 293–303 295
in 1 ml of distilled water. One hundred microliters of
the suspension was used to prepare 10-fold dilutions,
and 500 ml of each was plated on standard methods
agar (Pronadisa, Spain; composition (g/l): peptone
from casein 5.0; yeast extract 2.5; dextrose 1.0;
agar-agar 15.0) and incubated 24 h at 28 8C. Plates
with 30–40 colonies were selected and all bacterial
colonies growing on the surface were removed with
1.5 ml of 0.15 M citrate buffer (pH 4.0) by gently
scraping with a glass rod; this suspension was used
for PLFA analysis of the culturable microorganisms
(culturable PLFAs). The remaining volume (0.9 ml)
was used for PLFA analysis of total microorganisms
(total PLFAs).
Lipids were extracted as in Frostega
˚rd et al. (1993),
based on the method of Bligh and Dyer (1959) and
fractionated on silicic acid (100–200 mesh, Unisil)
columns by eluting with chloroform, acetone and
methanol. The polar lipids (containing phospholipids)
were subjected to a mild alkaline methanolysis (Dowl-
ing et al., 1986), which transformed the fatty acids of
the phospholipids into free fatty acid methyl esters.
These were analyzed by gas chromatography, accord-
ing to the methods described by Frostega
˚rd et al.
(1993). All solvents used were HRGC-grade.
Fatty acids were designated as the total number of
carbon atoms: number of double bonds, followed by
the position of the double bond from the methyl end
(o) of the molecule, and cand tindicate cis and trans
configurations, respectively. The prefixes aand i
indicate anteiso- and iso-branching, respectively; br
indicates an unknown methyl branching position,
10Me indicates a methyl group on the 10th carbon
atom from the carboxyl end of the molecule and cy
refers to cyclopropane fatty acids.
2.7. Nitrogen acquisition
Total nitrogen per gram of plant was determined
by colorimetry as in Smith (1980), after Kjeldahl
digestion in Prolabo Maxidigest-MX350 microwave
digester.
2.8. Biometric parameters
For shoot length and surface, plants were gently
pressed in filter papers. The analysis was carried out
using an image analyzer Delta-T System with DIAS
software. Plants were dried at 55 8C for several days
and dry weight was recorded. Shoot length, shoot
surface area and dry weight were determined.
2.9. Statistics
One-way analyses of variance (ANOVA) followed
by LSD tests (P<0:05) (Sokal and Rohlf, 1979) were
used to detect treatment differences on seedling
growth, nitrogen and chitin-ergosterol content. The
percentage of the PLFA values from rhizosphere and
bacterial suspension recovered from plates were log
10
transformed, prior to a principal component analysis
(PCA) (Harman, 1967) to elucidate the major varia-
tion patterns. Two PCAs were made, one for total
PLFAs and the other for culturable PLFAs. In each
case, the matrix subjected to multivariate analysis was
21 (7 treatments 3 sampling times) 21 (PLFAs
analyzed). The multivariate calculations, ANOVA
and LSD were performed with the computer program
SYSTAT
TM
v 5.05, for Windows
TM
.
3. Results
The inoculation of oak seedlings with B. licheni-
formis (B2) (Fig. 1) resulted in a significant increase in
all the parameters measured, not only when compared
with the control but also with other treatments, except
for dry weight under the influence of PtB2. Those
plants treated only with the fungus (Pt) did not show
significant differences with controls. In combined
treatments with fungus and bacteria, only PtB2 sig-
nificantly increased shoot dry weight.
Neither treatment increased nitrogen over the con-
trols (Fig. 2). However, nitrogen content in PtB1
(2.55 mg N/g plant) and Pt (1.44 mg N/g plant) was
significantly lower than in controls (4.92 mg N/g plant).
Ergosterol reflects the amount of live mycelia pre-
sent in treatments inoculated with the fungus (Fig. 3).
During the winter, all treatments showed reduced
levels of the metabolite. Ergosterol content was higher
in inoculated than in control plants during the vege-
tative growth period, although only Pt (6.22 mg/g root)
and PtB1 (5.78 mg/g root) showed significant
differences with controls. When the fungus was
co-inoculated with B. licheniformis (B2), ergosterol
concentration decreased significantly (1.5 mg/g root).
296 J. Domenech et al. / Forest Ecology and Management 194 (2004) 293–303
Fig. 1. Shoot length, shoot surface area and dry weight of inoculated plants and controls as calculated from the average of samples. Bars with
different letters indicate statistical differences in LSD. Lines at the top of each bar represent SD. Treatments: B. pumilus (B1), B. licheniformis
(B2), P. tinctorius (Pt), non-inoculated controls (C).
Fig. 2. Total nitrogen per plant of inoculated plants and controls as calculated from the average of samples. Bars with different letters indicate
statistical differences in LSD. Lines at the top of each bar represent SD. Treatments: B. pumilus (B1), B. licheniformis (B2), P. tinctorius (Pt),
non-inoculated controls (C).
J. Domenech et al. / Forest Ecology and Management 194 (2004) 293–303 297
Chitin content appears in Fig. 3. As ergosterol,
chitin was determined in the vegetative and winter
period. In both periods, differences between the con-
trol and the treatments were non-significant. However,
chitin levels increased in all treatments during the
winter period, coupled with a decrease in ergosterol
levels.
The PCA of total PLFAs partitioned from each
rhizosphere and sampling time resulted in a separation
along the first component. This accounts for 89.59% of
the variance (Fig. 4) which separates S3 from S1 and
S2 plots which are located at the highest values of axis
I. S3 plots appear at the lowest values of first principal
component. There are no differences between S1 and
S2 in the cenotic composition of treated plants, due to
the low percentage of variance absorbed by axis II
(9.54%). The principal loading factors for this group
are Me18, 18:0, 18:1o7(Fig. 4, underlined). When
analyzing S3, the composition of the rhizosphere is
completely different from either S1 or S2. The most
relevant fatty acids are i17:0, br17:0, cy19 and 18:1o9
(Fig. 4, underlined).
The culturable PLFAs obtained from plates repre-
sent only a fraction of the whole soil community
(Fig. 5). Scores of PCA show location of S1 at the
highest values of the first component and at the lowest
ones of the second axis. On the other side, S2 and S3
appear at the highest values of axis II and the lowest of
axis I. In this second group only PtB2 and B1 appear
slightly separated from the other treatments. This
analysis reveals a separation of the different plots along
the first two principal components, which explain
39.72 and 31.03% of the variance, respectively. In
addition, the control of S1 deviates substantially from
Fig. 3. Ergosterol and chitin analysis of inoculated plants and controls as calculated from the average of S1, S2 and S3 for the vegetative
period and S4 for the winter period. Bars with different letters indicate statistical differences in LSD. Lines at the top of each bar represent SD.
Treatments: B. pumilus (B1), B. licheniformis (B2), P. tinctorius (Pt), non-inoculated controls (C).
298 J. Domenech et al. / Forest Ecology and Management 194 (2004) 293–303
6
5
4
3
2
1
0
-1
-2
-3
-4
-20 -15 -10 -5 0 5
Axis I (89.59%)
Axis II (9.54%)
cy19
16:1w9
i:17
br17:0
B2
B1B2
PtB1
Pt
C
PtB2
B1
S1
S2
S3
B1
B2
C
PtB2
B1B2
Pt
Pt
PtB1
PtB1
B1
B1B2
C
PtB2
B2
Me18
18:0 18:1w7
Fig. 4. PCA showing variation in scores of total PLFAs and loading values for some individual PLFAs (underlined) of treated plants and
controls at the different sampling times. Treatments: B. pumilus (B1), B. licheniformis (B2), P. tinctorius (Pt), non-inoculated controls (C).
1402 64 8 10 12
6
-2
0
2
4
-2
8
10
Axis I (39.72%)
Axis II (31.03%)
17:1w8
Me17
i:16
18:1w9
B2
B1B2
PtB1
Pt
C
PtB2
S1
S2
S3
B1
B2
C
PtB2
B1B2
Pt
Pt
PtB1
PtB1
B1
B1B2
C
PtB2
B2
Me18
i:17
18:2w6
B1
16:1w7c
Fig. 5. PCA showing variation in scores of culturable PLFAs and loading values for some individual PLFAs (underlined) of treated plants and
controls at the different sampling times. Treatments: B. pumilus (B1), B. licheniformis (B2), P. tinctorius (Pt), non-inoculated controls(C).
J. Domenech et al. / Forest Ecology and Management 194 (2004) 293–303 299
the scores corresponding to inoculated plants. The fatty
acid components of culturable PLFAs with the highest
loading factors appear underlined in Fig. 5: 16:1o7c,
18:1o9 and i17:0 are close to S1, while Me:18, 17:1o8,
18:2o6, i16:0 and Me17 are related to S2 and S3.
4. Discussion
Inoculation with B. licheniformis resulted in
enhanced growth of oak seedling as reflected by
significant increases in shoot surface area, shoot
length and shoot dry weight. This increase is not
associated to changes in total microbial rhizosphere
communities after inoculation. Co-inoculation of B.
licheniformis and Pisolithus resulted in the inhibition
of mycorrhizal growth.
Chitin and ergosterol parameters have been deter-
mined in roots colonized by some ectomycorrhizal
species in forest plants (Frey et al., 1992; Ekbald et al.,
1998; Probanza et al., 2001). A component of mem-
branes, ergosterol, is considered an indicator of meta-
bolically active fungi (Nylund and Wallander, 1992).
The content of chitin, a component of the cell wall,
reflects all fungal biomass, living or dead, constituting
an indicator of fungal growth over the root systems.
During vegetative plant growth, Pt and PtB1 showed
significantly high levels of ergosterol (Fig. 3) over the
control. PtB2 and control had lowest amount of this
metabolite, suggesting that B. licheniformis (B2) inhi-
bits fungal growth. In the winter period, ergosterol
decreased to low levels. This decrease may be due to
environmental factors which determine the physiolo-
gical shutdown of the plant on one hand, and, on the
other hand, to the death of the active mycelium
surrounding roots (Lo
´pez et al., 2001).
During the vegetative period, differences in chitin
content between the control and the rest of the treat-
ments were non-significant (Fig. 3). In the winter
period, chitin content in PtB2 was significantly lower
than Pt, coinciding with the results obtained for
ergosterol in the vegetative period (Fig. 3). These data
support the hypothesis that B. licheniformis (B2) has a
negative effect on mycorrhization. The high levels of
chitin detected in the control may be due to a colo-
nization of the rhizosphere by autochthonous fungi or
to the presence of microarthropods in the rhizosphere
(Nylund and Wallander, 1992).
Our results showed that B. licheniformis (B2)
enhanced plant growth by itself, therefore, plant
growth promotion can be attributed to this strain
and not to mycorrhiza. These results were consistent
with others (Shishido et al., 1996a,b;Probanza et al.,
2001) who reported that growth promotion by Bacillus
strains on conifer seedlings was not dependent on
mycorrhizae. Previous reports show that one of the
mechanisms by which PGPRs stimulate seedling
growth is through phytohormone production (Holl
et al., 1988; Chanway, 1997). Our earlier findings
show that the two Bacillus strains are able to synthe-
size plant growth regulators (Gutie
´rrez-Man
˜ero et al.,
1996, 2001). Increases in shoot length over non-trea-
ted plants occurs via internodal increase, not by an
increase in the number of nodes (data not shown).
Hence, gibberellins are more likely to be responsible
for this effect rather than IAA-like compounds. The
increase in the growth parameters considered in plants
inoculated with bacteria seems to be the result of a
balanced development of photosynthetic apparatus,
which is coupled with an efficient system of nutrient
acquisition by the plant, as shown for N (Fig. 2).
Mycorrhiza may serve as a carbon and nitrogen sink
early on the interaction, revealing a complex relation-
ship between soil microflora and bacterium in nursery
and greenhouse settings (Gutie
´rrez-Man
˜ero et al.,
1996; Vonderwell and Enebak, 2000). Our results
show that N per plant was the lowest in those treat-
ments in which the mycorrhiza achieved a better
development according to ergosterol analysis, Pt
and PtB1. This supports the notion that the fungus
absorbs part of this N. However, B. licheniformis (B2)
and PtB2 show similar values in N content per plant
indicating the inhibiting effect of this bacterial strain
on Pt as pointed out above. Other studies in other
Quercus species indicate that inoculation with P.
tinctorius stimulated the uptake of N, P, and K, yet
no growth promotion was detected (Tam and Griffiths,
1993). On the other hand, and considering all growth
parameters together with N content, it may be con-
cluded that B. licheniformis stimulates N absorption
by the plant, since seedlings inoculated with this
bacteria show higher values in shoot surface area,
shoot length and dry weight than controls and similar
nitrogen content.
Inoculation with mycorrhizal fungus (Pt) did not
result in growth increase of oak seedlings. Moreover, a
300 J. Domenech et al. / Forest Ecology and Management 194 (2004) 293–303
positive long-term effect on mycorrhizal infection
cannot be ruled out. When B. licheniformis was co-
inoculated with P. tinctorius a strong inhibitory effect
on the fungus (Fig. 3) was observed. Bacterial inhibi-
tion of ectomycorrhiza could be a result of: (1)
bacterial niche exclusion on roots (Mehaffee, 1991);
(2) micro-scale nutrient and water competition in the
rhizosphere (Germida and Walley, 1996; Shishido and
Chanway, 1998); and/or (3) the production of anti-
fungal metabolites by bacteria (Barea et al., 1998).
Results from total PLFA analysis from the rhizo-
sphere appear in Fig. 4. Scores of sampling times S1 and
S2 plot together, indicating that the cenotic composition
of the rhizosphere is similar. A separation of the control
from all inoculated treatments could be expected at S1,
though it only occurred in Pt. This could indicate loss of
inoculum, but this possibility may be discarded in view
of the different evolution of the results in the following
sampling times. Therefore, it may be explained because
the background of the whole rhizosphere community
would be masking the inocula. This hypothesis coin-
cides with the results obtained for the principal loading
factors, since only Me18 is related to fungal markers
(Griffiths et al., 1998) and 18:1o7 to Gram-negative
bacteria. In S3, plots are situated around the most
negative values on axis I, indicating some differences
in the cenotic composition during the experiment. This
may be explained by successional changes as there are
no differences among treatments. For the second group,
S3, the fatty acids br17 and i17 are related to Gram-
positive bacteria (Griffiths et al., 1998) as indicators of
the Gram-positive bacterial inocula. As in S1 and S2,
some fatty acids characteristic of Gram-negative bac-
teria are also present (Steer and Harris, 2000). Other
studies have shown that growth promotion is linked to
low perturbation of the rhizosphere system following
inoculation (Ramos et al., 2002). Although none of the
inoculum severely altered the composition of the rhizo-
sphere microbial communities, only B. licheniformis
stimulated oak growth. Moreover, other authors have
shown that although maintaining the equilibrium of the
original rhizosphere community structure is important
to improve plant growth, it is not the only condition to
allow the beneficial effect (Ramos et al., 2003).
The PCA of plates reflect the viable and culturable
bacterial populations which were verydifferent between
S1 and the others. In S1, the control separates from all
other treatments, probably due to the alteration of the
culturable rhizosphere community by the different
inocula. The principal loading factors in this group
are 18:1o9 and 16:1o7c, characteristic of fungi (Olsson
et al., 1995), and i17, of Gram-positive bacteria (Zelles,
1999), correspondingto the inoculated bacilli and fungi.
The viable and culturable population in S2 and S3 was
very similar. The most significant loading factors for
this second group were 18:2o6, Me18 and Me17 related
to fungi (Wander et al., 1995; Zelles, 1999), and i16:0
described as a biomarker of Gram-positive bacteria
(Ibekwe and Kennedy, 1999). These results are in
accordance with the inoculated treatments.
Analyses of total and culturable PLFAs show dif-
ferent behavior. Culturable PLFAs show a similar
cenotic structure in S2 and S3, revealing that the
inocula disappeared within 60 days. This disappear-
ance, together with the high dispersion of the samples
and the variance absorbed by the axis, indicate the
greater dynamism of viable and culturable populations
and their prevalence in the rhizosphere system, as
described by Ba
˚a
˚th (1994). Although the inocula
disappeared within 60 days after inoculation, growth
parameters were measured in S4 (270 days) and, at
that time, the effect of the inocula persisted (Fig. 1).
We conclude that B. licheniformis promotes Q. ilex
seedling growth and stimulates the incorporation of N.
This biological effect does not imply a synergic effect
with mycorrhizal infection. Furthermore, this strain
inhibited fungal growth, although a positive response
of mycorrhiza in a long-term experiment cannot be
discarded. The introduction of inocula affects the
microbial rhizosphere composition of the total commu-
nity and the culturable populations in different ways.
Acknowledgements
This research was supported by the Comunidad
Auto
´noma de Madrid (CAM), Research Project
06M-031-96. JD was formerly a postdoctoral fellow
of CAM. We also thank Linda Hamalainen for editorial
help.
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