Agriculture, Ecosystems and Environment 103 (2004) 405–411

Arbuscular mycorrhiza and enzymatic activities in the rhizosphere

of Trachypogon plumosus Ness. in three acid savanna soils
J.C. López-Gutiérrez∗ , M. Toro, D. López-Hernández
Facultad de Ciencias, Laboratorio de Estudios Ambientales, Instituto de Zoologı́a Tropical, Universidad Central de Venezuela,
Caracas 1041-A, Apartado 47.058, Venezuela

Abstract
Phosphorus (P) is one of the most limiting macronutrients in savannas. Seasonality strongly affects nutrition processes in
such ecosystems. We studied arbuscular mycorrhizae (AM), microbial and phosphatase activity in the rhizosphere of Trachy-
pogon plumosus Ness. in three acid savanna soils differing in taxonomic order and P content. Microbial number and activity, P
mineralization and AM dynamics were quantified in rhizospheric samples of T. plumosus during the dry and the rainy season
at three sites in the Estación Experimental La Iguana, Guárico State, Venezuela. Soils were characterized and infective AM
propagules enumerated using the most probable number method on Sorghum vulgare Pers. Acid phosphatase activity (APA),
dehydrogenase activity (DHA) and number of bacteria and fungi were determined in rhizospheric samples. AM colonized root
length percentage (%CRL) was also determined in T. plumosus. Our data showed that the three soil orders had a very low fertil-
ity. AM infective potential showed values similar to those reported for tropical savannas. Also, AM %CRL was high for a grass
species. APA increased in the rainy season in all cases. However, DHA and microbial counts decreased during the rainy season
suggesting that soil microorganisms do not mediate the increase in APA. AM colonization, seasonal changes in microbial
activity and in APA seemed to be important processes for P availability and T. plumosus P-uptake in savanna ecosystems.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Phosphatase; Savanna; Dehydrogenase; Arbuscular mycorrhiza; Trachypogon

1. Introduction dry and rainy seasons. Most of Venezuelan savanna

Nearly one third of the Venezuelan territory is dom-
inated by savanna ecosystems. In general savanna soils
are highly weathered, acidic, with a very low fertility
which results in a low nutrient content and low primary
productivity (Jordan, 1984). Phosphorus (P) is one of
the most limiting macronutrients in savannas due to
the high weathering rate of these soils (Tiessen et al.,
1984). These ecosystems are subject to very marked
∗ Corresponding author. Present address: Institute National de la
Recherche Agronomique, UMR 1229, Microbiologie et Géochimie
des Sols, 17 rue Sully, B.V. 86510. 21065 Dijon, France; Tel:
+33-3-80-69-30-90; fax: +33-3-80-69-32-24.
E-mail address: jclopez@alum.wustl.edu (J.C. López-Gutiérrez).
ecosystems are dominated by grasses such as Tra-
chypogon plumosus Ness., in fact some savannas are
known as Trachypogon savannas. Other shrub and tree
like species, such as Curatella americana Linn., Mau-
ritia flexuosa Linn. and Bowdichia virgiloides H.B.
and K. can be found, however, they do not form a
continuous canopy (Sarmiento, 1981).
In a detailed P fractionation study in Venezuelan
savannas Hernández-Valencia and López-Hernández
(1999) show that only 4% of the total soil P is either
available or labile inorganic P (Pi). This fractionation
also shows the importance of soil microorganisms
in P-cycling, since 6% of the phosphorus in the
ecosystem is present as microbial P. Finally, 24% of
the soil P is labile and moderately labile organic P

0167-8809/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.agee.2003.12.011
406 J.C. López-Gutiérrez et al. / Agriculture, Ecosystems and Environment 103 (2004) 405–411
(Po). Even though Po is not readily available for plant
uptake, it can become available after mineralization to
inorganic forms (Ascencio, 1997). Acid phosphatase
activity (APA) allows the use of less readily avail-
able P forms hydrolyzing Po to Pi (Clarholm, 1993).
Most of the Po mineralizing activity occurs at the
rhizosphere where phosphatases released from plant
roots (Helal and Dressler, 1989), soil microorganisms
(Asmar et al., 1995) and earthworms (Satchell and
Martin, 1984) are present.
Arbuscular mycorrhizae (AM) contribute to plant
growth and nutrient uptake, specially P (Smith and
Gianinazzi-Pearson, 1988). Contribution can be a
direct effect through an exploration of a larger
volume of soil and/or a hyphal access to soil
P pools not available to plants (Koide, 1991;
Li et al., 1991; Jayachandran et al., 1992). Some
authors even assert that AM mycelia may have P min-
eralizing activity (Jayachandran et al., 1992; Tarafdar
and Marshner, 1994).
APA (Fox and Comerford, 1992; Clarholm, 1993)
as well as AM colonization (Tarafdar and Marshner,
1994; Joner and Jakobsen, 1995) can be induced by
P deficiency. Furthermore, rhizospheric microorgan-
isms and AM can synergistically contribute to plant P
nutrition (Toro et al., 1996; Singh and Kapoor, 1998).
We hypothesize that some specific mechanisms
must be operating in the plant rhizosphere to allow
plant growth in savannas. Thus, AM, soil microor-
ganisms and APA in the rhizosphere are considered
as crucial mechanisms for P availability and plant
P-uptake. Our interest focuses on the biochemical
processes occurring in the rhizosphere of native plant
species of P-deficient soils. Therefore microbial activ-
ity, as measured by viable plate counts and dehydro-
genase activity (DHA), mineralization activity (APA)
and AM dynamics was studied in the rhizosphere of
T. plumosus growing on three seasonal savanna soils
differing in phosphorus content and soil order.
2. Materials and methods
2.1. Sampling sites
Sampling sites were located in savannas with T.
plumosus Ness., as the dominant herbaceous compo-
nent, and isolated C. americana Linn., as the tree
component, in Estación Experimental La Iguana near
Santa Marı́a de Ipire, Guárico State, in northeastern
Venezuela (8◦ 25 N and 65◦ 25 W). The average annual
precipitation is 1200 mm (concentrated in a rainy sea-
son between May and September), and the average
monthly temperature is 27.9 ◦ C.
2.2. Soil sampling and characterization
The sampling sites correspond to an Entisol, an
Ultisol and a Vertisol that differ in texture. For the gen-
eral soil characterization air-dried 2 mm sieved sam-
ples from the dry season were used. For the rest of the
biological analysis soil samples from the rhizosphere
of T. plumosus individuals, as well as roots, were col-
lected in the middle of the dry and the rainy seasons
and stored in sealed plastic bags at 4 ◦ C until used.
Soil characterization involved: (1) Pt determined
after perchloric/sulfuric acid digestion and colorimet-
ric P determination (Murphy and Riley, 1962; Olsen
and Sommers, 1982); (2) P-NaHCO3 determined
with 0.5 M NaHCO3 extraction after 16 h shaking
(Bowman and Cole, 1978); (3) soil pH measured with
a glass electrode (1:5 soil:water ratio) and soil texture
with the Bouyoucos method.
2.3. Rhizospheric enzymatic determinations
DHA was determined colorimetrically as the degra-
dation of triphenyl tetrazolium chloride (TTC) to
triphenyl tetrazolium formazan (TTF) after 24 h of
1.5 g fresh soil sample incubation. Values are ex-
pressed as microgram of TTF released per gram of
dry soil per 24 h, according to Casida et al. (1964).
APA was determined colorimetrically as the degra-
dation of PNP-P (p-nitrophenyl phosphate) to PNP
(p-nitrophenol) after a 30 min incubation of 1 g fresh
soil sample at pH 4.5. Values are expressed as micro-
gram of PNP released per gram of dry soil per hour,
according to Tabatabai and Brenmer (1969).
2.4. Rhizospheric bacterial and fungal
viable plate counts
Bacterial and fungal colony forming units (CFUs)
were counted using the standard dilution plate tech-
nique of fresh soil suspension on selective media.
 J.C. López-Gutiérrez et al. / Agriculture, Ecosystems and Environment 103 (2004) 405–411
Bacteria were determined on tryptic soy agar (Martin,
1975). Fungi were estimated on rose bengal agar
(Martin, 1950). Colonies were counted on the ap-
propriate dilutions after 2 and 5 days, respectively,
incubation at 25 ◦ C. The results are reported as log of
bacterial or fungal CFU per gram of dry soil.
2.5. Mycorrhizal determinations
AM infective potential was determined by the
most probable number (MPN) of infective propagules
method according to Sieverding (1991). An amount
of 5 kg of native soil (0–15 cm) collected from each
site in the dry season was sieved through a 1 cm mesh
and 1 h steam sterilized for three consecutive days.
The 500 g pots were prepared using sterile/native
soil serial dilutions in 1:10 proportion. One seed per
pot (10% sodium hypochlorite surface sterilized) of
Sorghum vulgare Pers. was placed and after 40 days
roots were cleared and stained as described below to
observe presence or absence of AM structures. Data
is expressed as number of AM propagules in 100 g
dry soil and the confidence limits assigned according
to Fisher and Yates (1970).
To determine AM colonization, T. plumosus root
samples collected in the rainy and the dry season
were carefully rinsed, cleared and trypan blue stained
(Phillips and Hayman, 1970). AM colonization in
these samples was quantified in a microscope at 10×
magnification and expressed as percentage of colo-
nized root length (%CRL), according to Giovanetti
and Mosse (1980).
2.6. Statistical analyses
Data were analyzed with one way ANOVA test to
observe statistical differences between media of sam-
Table 1
General soil characterization and most probable number of three acid savanna soils
Soil order Entisol
Texture Sandy
pH 5.4 a
P-total (␮g g−1 ) 59.8 a
P-NaHCO3 (␮g g−1 ) 4.9 b
AM infective potential propagules per 435.4 (100.2–456.0)
100 g soil (95% confidence limits)
Values followed by the same letter do not differ significantly. Duncan’s test (P = 0.05).
407
ples. When media were different a Duncan’s test was
applied to observe statistical differences (P = 0.05).
Parameters for the soil characterization were com-
pared between soils while the other parameters were
compared only between seasons for each soil order.
3. Results and discussion
3.1. Soil chemical and textural analyses:
phosphorus forms
Soils were slightly acid and as expected they had a
very low Pt content (Table 1). P-NaHCO3 , which is
considered to be plant available (Bowman and Cole,
1978), is extremely low in all three soils. This result
supports findings for other seasonal tropical ecosys-
tems were P is one of the most limiting macronutrients
(Roy and Singh, 1995).
In general, available P seem to be higher as
texture becomes finer (O’Halloran et al., 1987;
López-Hernández and Niño, 1993; Zubillaga and
Giuffré, 1999), our results showed exactly the oppo-
site, P-NaHCO3 was lower as texture became finer,
being extremely low in the case of the Vertisol. P
adsorption in these soils is higher as silt and clay con-
tent increase (O’Halloran et al., 1987). The Vertisol in
this study differs from others in the world because of
the acid pH instead of the basic pH of those derived
from calcareous material which are very fertile soils
(USDA, 1978).
3.2. Enzymatic results and viable plate counts
APA significantly increased during the rainy season
in all soil orders. This increase, however, was propor-
tionally higher for the Entisol (Fig. 1). These results
Vertisol Ultisol
Clay loam Sandy clay loam
5.7 b 5.4 a
143.5 c 93.0 b
0.75 a 1.4 ab
435.4 (100.2–456.0) 189.5 (44.4–201.84)
408 J.C. López-Gutiérrez et al. / Agriculture, Ecosystems and Environment 103 (2004) 405–411

250,00 3,5
b a
3 a a
200,00 b b a
log fungal cfu / g

a
µg PNP/g h

2,5
150,00 Dry
a 2
Dry
100,00 b a Rainy
Rainy
1,5
50,00
a 1
0,00 0,5
Entisol Vertisol Ultisol
0
Soil Order Entisol Vertisol Ultisol

Soil Order
Fig. 1. Seasonal changes in acid phosphatase activity in the rhi-
zosphere of T. plumosus in three acid savanna soils (mean ± S.D.,
Fig. 3. Seasonal changes in viable fungal plate counts in the rhi-
n = 3). Values followed by the same letter do not differ signifi-
zosphere of T. plumosus in three acid savanna soils (mean ± S.D.,
cantly. Duncan’s test (P = 0.05).
n = 3). Values followed by the same letter do not differ signifi-
cantly. Duncan’s test (P = 0.05).

suggest that P mineralization, as expressed by APA,

increases during the rainy season, when plant nutri-
ent uptake and nutrient leaching are higher (Roy and
Singh, 1995).
Dehydrogenases are not expected to be free in
soil, therefore, DHA expresses living cell activity and
that is why it is an indicator of biological activity
(Frankenberger and Dick, 1983). DHA significantly
decreased during the rainy season in all soil orders
(Fig. 2). Even though several soil factors such as
soil texture affect DHA, this enzymatic activity can
be used to detect soil seasonal changes (Cooper and
Warman, 1997). Furthermore, DHA can correlate with
changes in the microbial biomass due to long-term
soil amendment (Goyal et al., 1993).
In an attempt to relate DHA with another indicator
of soil microbial activity we quantified soil fungi and
250,00
bacteria. Viable plate counts are not expected to show
the same pattern as enzyme activities because selec-
tive media recovers only a proportion of soil microor-
ganisms and might include those in dormant phase
(Frankenberger and Dick, 1983; Riis et al., 1998).
The results showed that viable fungal counts (Fig. 3)
were significantly different between seasons only for
the Entisol, being higher during the rainy season.
Bacterial counts (Fig. 4) significantly increased dur-
ing the rainy season for the Ultisol, but decreased for
the Entisol and the Vertisol during the rainy season.
The latter result agrees with the microbial activity, as
expressed by DHA, significantly decreasing during
the rainy season at all sites (Fig. 2).
A higher bacterial count and DHA during the dry
season support the idea that in seasonal ecosystems
microbial grazers and plants are not as active during

8
200,00 b b
b
µg TTF/ g 24h

7
log bacterial cfu / g

b a
150,00 6 a

Dry 5 Dry
a
100,00 4 Rainy
b Rainy
3
50,00 2
a
b a a 1
0,00
0
Entisol Vertisol Ultisol Entisol Vertisol Ultisol
Soil Order Soil Order

Fig. 2. Seasonal changes in dehydrogenase activity in the rhizo- Fig. 4. Seasonal changes in viable bacterial plate counts in the rhi-
sphere of T. plumosus in three acid savanna soils (mean ± S.D., zosphere of T. plumosus in three acid savanna soils (mean ± S.D.,
n = 3). Values followed by the same letter do not differ signifi- n = 3). Values followed by the same letter do not differ signifi-
cantly. Duncan’s test (P = 0.05). cantly. Duncan’s test (P = 0.05).
 J.C. López-Gutiérrez et al. / Agriculture, Ecosystems and Environment 103 (2004) 405–411
the dry season while soil microorganisms are (Singh
et al., 1989). In fact, Ingham et al. (1986) showed how
in a seasonally dry ecosystem protozoa, fungivorous
microarthropods and bacterial feeding nematode pop-
ulations decrease while bacterial populations increase
during the dry season.
In savannas (Singh et al., 1991), as well as in
other tropical ecosystems affected by low fertility and
strong seasonality (Singh et al., 1989; Campo et al.,
1998), soil microorganisms, besides being adapted to
dry conditions, play an important role in the seasonal
P-cycling by immobilizing P during the dry season
and releasing P during the rainy season. The impor-
tance of soil microorganisms in these studies has
been evaluated in terms of the content of nutrients in
the microbial biomass. Our results confirmed these
earlier findings taking into account viable counts and
activity of soil microorganisms (DHA), instead of the
nutrient content present in the living cells or microbial
biomass.
Furthermore, microorganisms in the rhizosphere
may contribute to P nutrition through the synthesis
and release of phosphatases when P is not available
(Speir and Cowling, 1991; Clarholm, 1993). The de-
crease in DHA and bacterial counts during the rainy
season, however, suggests that the increase in APA
during that period is not mediated by rhizospheric
microorganisms, i.e., soil extracellular phosphatases
may have been already present in the soil or have
plant root origin.
3.3. Mycorrhizal status
The number of AM infective propagules (MPN)
was high and not significantly different in all three
soils (Table 1). Textural differences do not affect this
parameter, as suggested by Sieverding (1991) in nat-
ural ecosystems. The values were higher than those
reported in natural Colombian savannas, however, a
well-managed cassava (Manihot esculenta Crantz.)
crop may have values six-fold higher than natural
savannas (Sieverding, 1991). One of the reasons for
not finding significant differences in this parameter
is the fact that MPN method has wide confidence
limits (Porter, 1979). Moreover, this method involves
soil sample mixing and dilution which causes hy-
phal network rupture, yet it allows robust propagule
enumeration in soil (Jasper et al., 1993). Proper
409
100
90
% Colonized root length
b
80 a
a
70 b
60 a
a Dry
50
40 Rainy
30
20
10
0
Entisol Vertisol Ultisol
Soil Order
Fig. 5. Seasonal changes in AM colonization of T. plumosus in
three acid savanna soils (mean ± S.D., n = 3). Values followed by
the same letter do not differ significantly. Duncan’s test (P = 0.05).
agricultural management of savanna soil may increase
the soil AM potential, increasing the efficiency of
plant AM symbiosis for P-uptake (Dodd et al., 1990).
Considering that herbaceous plants with rapidly
growing fine roots and abundant root hairs are less
responsive to AM colonization (St. John, 1980), T.
plumosus showed rather high %CRL (Fig. 5). Some
authors (St. John et al., 1983; St. John and Uhl, 1983)
observed CRL of 54.7% for Panicum pilosum Sw. and
29.8% for Andropogon bicornis Linn., in savannas.
Even though both are grass species their %CRL is not
as high as our results which suggests that AM may
be a crucial mechanism for T. plumosus P-uptake in
savanna ecosystems.
AM colonization, registered as %CRL in T. plumo-
sus, was higher in the Entisol and Vertisol during the
dry season, but not in the Ultisol (Fig. 5). Several
environmental factors, seasonality being one of them,
may cause differences in %CRL. Such variations may
depend upon the specific structural, developmental
and physiological characteristics of the plant–AM
combination (Smith and Read, 1997). Furthermore,
the ability of the mycorrhiza to absorb nutrients is
linked to the development of external mycelium and
the proportion of the fungus which is metabolically
active (Hepper, 1981) which might not be reflected
on the %CRL (Tisserant et al., 1993).
On the other hand, Nelsen and Safir (1982) showed
how drought stress, even in fertile soils, causes high
levels of AM colonization because low moisture levels
can reduce nutrient diffusion rate, such as P, decreas-
ing plant availability. Accordingly, our results showed
410 J.C. López-Gutiérrez et al. / Agriculture, Ecosystems and Environment 103 (2004) 405–411
that, at least for the Entisol and the Vertisol, AM col-
onization (Fig. 5) was higher during the dry season,
when P mineralization (Fig. 1) was lower and micro-
bial immobilization (Figs. 2 and 4) was higher. Thus,
AM colonization was higher when P was less avail-
able for plant uptake.
4. Conclusions
Linderman (1988) coined the term mycorrhizo-
sphere and hypothesized that rhizobacteria and AM
fungi function in concert to promote plant growth. In
fact, specific systems of plant, AM and rhizobacte-
ria have been described (Andrade et al., 1997; Toro
et al., 1997). In the present study, on the other hand,
a natural system is described in which P mineraliza-
tion, rhizobacterial community and AM colonization
seem to be working in asynchrony to overcome the P
limitations set by seasonality. In savannas, as well as
in tropical ecosystems affected by seasonality, asyn-
chrony in such processes in the mycorrhizosphere
might be essential for plant P-uptake.
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