315

Plant and Soil 183:315-322, 1996.

@ 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Diversity patterns of arbuscular mycorrhizal fungi associated with cacao in

Venezuela
Gisela Cuenca and Erasmo Meneses
Venezuelan lnstitute fin" Scientific Resealz'h, Ecology Centel: P.O. Box 21.827, Caracas 1020-A. Venezuela *
Received 22 September 1995. Accepted in revised form 31 May 1996

Key words: diversity, spores, tree crop, tropics, VA-mycorrhiza

Abstract
The arbuscular mycorrhizal fungal (AMF) communities associated with cacao in Venezuela were studied. The
species of AMF spores present in sixteen cacao plantations and in one nursery were isolated and identified
when possible. The spore densities, species richness, diversity, Shannon-Wiener diversity index and dominance
concentration index for the AMF communities were calculated. Acaulospora scrobiculata was associated with
cacao plants in all study sites. No Scutellospora spp. were found in the analyzed soils. The spore number found in
cacao plantations was relatively lower as compared with other tropical crops (38 spores 100 g-1 soil up to 1674).
Soils that were cultivated with cacao for more than 40 years showed the lowest spore numbers. Species richness
and diversity of AMF communities associated with cacao, were negatively correlated with available P in soils.
The Shannon-Wiener diversity index was positively correlated with soil organic matter. These results indicate that
the traditional cacao cultivation practices used in Venezuela, maintain mycorrhizal infection on cacao plants. The
diversity of the AMF community is similar to that found in natural undisturbed ecosystems from Venezuela.

Introduction
Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with most economically important
crop plants. These fungi can improve plant growth
under low fertility conditions, confer tolerance to some
pathogens, improve the water balance of the plants and
contribute to the formation of soil structure (Jeffries
and Barea, 1994).
AMF species are usually considered as not having
any specificity towards different plant taxa as potential hosts (Harley and Smith, 1983). However, some
authors have found that plant species can influence the
species composition of AMF communities (Dodd et
al., 1990; Johnson et al., 1991, 1992). Also, it has been
suggested that monoculture may reduce the spectrum
of fungal species found in the soil after several years
of continuous cultivation (Allen and Boosalis, 1983;
Sieverding, 1991). Other authors have noticed that the
diversity of AMF communities tends to diminish when

* FAX No: + 5825041088

natural ecosystems are transformed in agroecosystems

(Sieverding, 1991; Siqueira et al., 1989).
Cacao (Theobroma cacao L.) in Venezuela is a traditional crop which is grown under the shade of some
trees, mainly legumes of the Erythrina genus. The litter
produced by these trees has beneficial effect on cacao
cultivation (Aranguren et al., 1982). Cacao is grown
in the soils which are relatively rich in nutrients. It
is grown in tropical humid climate with two seasons
through the year: the wet season usually from May
to November and the dry season from December to
April. Mean annual precipitation varies between 10002500 mm and annual temperature averages 26 C. The
management of this crop in Venezuela is done with a
low input technology and farmers usually do not apply
agrochemicals.
Cacao forms arbuscular mycorrhizae (Laycock,
1945). In Malaysia and Brazil an increase in the growth
of cacao seedlings in nursery conditions was obtained
by introducing AMF (Azizah-Chulan and Ragu, 1986;
Ezeta and Santos, 1981). In Venezuela the AMF isolates indigenous to a nursery soil were the most effi-

316
cient in promoting cacao seedling growth (Cuenca et
al., 1990). The aim of this work was to study the density
and diversity of AMF spores associated with cacao in
Venezuela. Additionally to the information that gives
us the frequency of occurrence of a particular AMF
species and the richness of species, diversity indexes
let us compare the number of spore types found in communities with different number of spores. There are not
many data in the literature in which these indexes have
been used to measure the diversity of AMF spores
present in a particular soil. Also we intend to study the
relationship between the diversity of AMF spores and
the age of cacao plantations and some edaphic factors.

and Riley (1962) and exchangeable Ca, K and Mg was

measured by atomic absorption spectrophotometry in
the same extract. Data presented are the mean values
of the three samples from each plantation studied.

Materials and methods

Arbuscular-mycorrhizal fungi communities infield

soils

Mycorrhizal infection of field samples

In each plantation studied, fine roots (< 3 mm diameter)
from 3-5 individuals were collected. Roots were taken
from 0-10 cm depth fixed in FAA, and stained in trypan
blue according to Phillips and Hayman (1970). Percentage infection was assessed by the gridline method
of Giovannetti and Mosse (1980). A minimum of 100
intercepts were scored for each sample.

Study site
Sixteen cacao plantations and one nursery from northern Venezuela were studied. Plantations sampled
included the main cacao-producing areas in Aragua,
Miranda and Sucre States. All the cacao plantations
surveyed were established under shade trees, such
as Erythrina poeppigiana (Leguminosae), Tabebuia
pentaphylla (Bignoniaceae), P ersea americana (Lauraceae) and Artocarpus altilis (Moraceae) (Table 1).
Farmers also use several species of Musa as a temporary shade plant and as their main crop until cacao
seedlings reach maturity (2-4 years). The sampled
plantations were of different ages (< 5 years up to > 70
years). The exact age of each plantation studied was
either unknown or based on information from farmers.
Therefore, in order to study the relationship of AMF
communities with age, plantations were classified as
young (< 20 years old), mature (20-30 years old) and
old (40-70 years old). Evaluation of the relationships
between plantation age, spore number and diversity
did not include the fertilized plantations (those marked
in Table 1 with a *).

Soil chemical analyses

From each plantation soil a subsample was air dried,
sieved (< 2ram) and pH was measured in water (1:5)
and in KC1 (1:5). Organic matter was assessed using the
method of Walkey-Black according to Jackson (1976)
and total nitrogen was measured by micro-Kjeldahl
procedure. Extractable P was assessed according to
Olsen et al. (1954) using sodium bicarbonate. In this
extract, P was measured by the method of Murphy

To study the AMF communities, three composite samples from the surface soil (0-20 cm) were taken at
random from each plantation during the months of
maximum rain (June-July). Only one nursery was sampled, but the spores were isolated from several different seedling cohorts. Soil samples were maintained in
closed plastic bags at 4 C until processing, no later than 15 days after collected. In the laboratory, the
samples were carefully homogenized and the AMF
spores were isolated from 50 g soil by the wet sieving
and decanting method followed by sucrose centrifugation (Sieverding, 1991). The isolated spores that
appeared healthy and without microbial attack were
counted and separated using a dissecting microscope
(60 x) according to their different types. Morphologically distinct spores were checked again using a compound microscope. From each different morphological
type, a permanent slide was prepared using polyvinyllactoglycerol alcohol (PVLG) or PVLG + Melzer's
Reagent, as a mounting medium. The specimens were
identified to genus and, when possible, to species. For
calculation purposes we considered sporocarps (Sclerocystis or loose multispore groups) as only one unit
to count. Voucher specimens are available (numbers:
Cuenca 40 to 63 and Cuenca 87 to 142) and can be sent
upon request to the authors.
With the data obtained we calculated the richness,
diversity, Shannon-Wiener diversity index and dominance concentration for each sampled site according to
the following equations:
Richness: R = Number of species found in a sample.
Diversity:

D : S/logN

317

Table 1. General description of the cacao plantations studied in this work

N
I
2
3
4a

5a

Site

Aprox. age

Shaded by

Management

Boca de Caucagua.
Barlovento, Miranda State
Boca de Caucagua,
Barlovento, Miranda Stale
Tapipa Grande,
Barlovento, Miranda State
Ocumare de la Costa,
Hacienda Monasterio,
Aragua State
Ocumare de la Costa.
Hacienda Monasterio,
Aragua State

30 years

Cupravit b and prunning

2 years

Erythrina poeppigiana,
Artocarpus altilis, Musa sp.
Ecythrina poeppigiana,
Artocalpus altilis, Musa sp.
Erythrina poeppigiana,
Artocarpus altilis, Musa sp.
EiTthrina poeppigiana. Musa sp.

> 20 years

Erythrina poeppigiana. Musa sp.

30 years
>70 years

6~'

Ocumare de la Costa,
Hacienda Monasterio.
Aragua State

> 30 years

Eiythrina poeppigiana. Musa sp.

La Esmeralda. Ocumare de
la Costa, Aragua State
Santa Cruz de la Vega,
Aragua State
Ri'o Seco, Yaguaraparo,
Sucre State
Rio Seco, Yaguaraparo,
Sucre State

> 70 years

Tabebuia pentaphylla, Ervthrina

poeppigiana
Etythrina poeppigiana

8a
9
10a

5 years
40-50
years
> 30 years

Persea americana, Citrus sp.,

Erytkrina poeppigiana
Erthrina poeppigiana. Spondias
mombin, Persea americana, Musa

3M years

MangiJk,ra indica. Persea

americana, Citrus sp.
Ecvthrina poeppigiana, Musa sp.,
Persea americana
Eo'thrina poeppigiana, Mangifkra
indiea, Persea anterieana
Persea americana, E13,thrina
poeppigiana, Musa sp.
Tabebuia pentaphylla, Persea
antericana, Et3,thrina poeppigiana
lnga sp., Spondias mombin
Erytkrina poeppigiana
lnga sp., Spondias mombin
Ervthrina poeppigiana

sp.
11
12
13
14
15
16
Nutsery

Los Palmares,
Yaguaraparo, Sucre State
Rio Seco, Yaguaraparo
Sucre State
Rio Seen, Yaguaraparo,
Sucre State
Ri'o Seco Yaguaraparo.
Sucre State
Yaguaraparo, Sucre State
Loma Maffn, San Jos6.
Sucre State
Panaquire. Miranda Stale

> 30 years
> 30 years
4-5 years
6 years
30-40
years
0-6 months

Cupravit and pruning

No management
Fertilization at
transplanting
(12:24:12:) 1 kffplant
Each year
fertilization with
12:24:12:1 kg/plant
Each year
fertilization with
15:15:15:1 kg/plant
No management
Fertilized with
15:15:15:1 k~plant
No management
Cupravit, it was
fertilized 6 years
ago (15:15:15:)
No management
No management
No management
No management
No management
No management
Cupravit

a Fertilized plantations excluded from the analysis of the influence of age on AMF community.
b Cupravit: Copper oxychloride applied to cacao leaves with phytosanitary purposes, once in a year.

W h e r e S = total n u m b e r o f s p e c i e s in t h e s a m p l e , a n d
N = total n u m b e r o f s p o r e s in t h e s a m p l e ( W h i t t a k e r ,
1975).

Dominance Concentration:
(Simpson's index)

W h e r e C is t h e i n d e x n u m b e r , s is t h e total n u m b e r
o f s p e c i e s in t h e s a m p l e , a n d p is t h e p r o p o r t i o n o f all
s p o r e s in t h e s a m p l e that b e l o n g to s p e c i e s i ( B a r b o u r
et al., 1987).

C = y~(pi) 2
i=l

S h a n n o n - W i e n e r D i v e r s i t y I n d e x H I ---- - ~
i=l

pi In pi

318
Where H' is thought to represent the uncertainty or
"information" of the community. The more variable
its composition, the more uncertain and unpredictable
each sample of it would be (Barbour et al., 1987).

Root inoculation experiment

To be certain that particular fungal species were colonizing cacao roots, fine cacao roots from the five plantations in Aragua State (Numbers 4, 5, 6, 7 and 8) were
collected and used to inoculate Vigna luteola seedlings.
The roots collected from three different cacao individuals in each plantation were carefully washed with tap
water and sonicated for 5 min to eliminate soil particles. Clean roots were cut into 2-cm pieces and used as
inoculum for pregerminated seeds of V. luteola, which
were sown in 1 kg pots (three/site) containing sterilized soil collected from Ocumare plantations. A treatment without root inoculum was also included. Three
months after, sowing, a soil sample (50 g) from each
pot was used to isolate AMF spores by wet sieving,
decanting and sucrose centrifugation.

Statistics
Spore counts were logarithmically (log 10) transformed prior to statistical analysis. Variances were
compared by one-way ANOVA followed by Tukey's
test (p < 0.05). Spearman rank correlation between
calculated indexes, spore populations from individual species and edaphic characteristics were calculated
according to Siegel (1978).

the sites (Figure 1). We did not find any Scutellospora

species and only one species of Gigaspora (very similar to G. albida Schenck and Smith) in the plantations
studied. In contrast, seven Acaulospora species were
associated with cacao plantations.
Diversity of AMF communities did not vary with
plantation age (Table 3). None of the measured indexes (Richness, Diversity index, Shannon-Wiener index,
or Simpson's dominance concentration) varied significantly with age (Table 3). However, there was a significant negative correlation between richness, diversity
(calculated according to Whittaker) and the available
P in soils (Table 4). Also the Shannon-Wiener index
was positively correlated with the content of organic
matter of the soil, and Simpson's Dominance Concentration was negatively correlated with soil organic
matter (Table 4). The number of spores of Glomus sp 1
resulted positively correlated with available P, Mg and
K in soils. In contrast the number of Glomus sp2 and
GIomus sp3 spores resulted negatively correlated with
available P in soils. Glomus sp2 also showed a negative
correlation with K in soils.
The root inoculation experiment with V. luteola as a
trap plant showed that Acaulospora scrobiculata, Scle~)cystis sinuosa Gerdermann and Bakshi, Acaulospora
mellea Spain and Schenck and an unidentified Glomus
species (Glomus sp3) formed mycorrhizal associations
with cacao roots. The non-mycorrhizal control plants
were significantly smaller than the V. luteola plants
inoculated with cacao roots.

Discussion
Results
The chemical analyses of the plantation soils (Table 2)
show that cacao in Venezuela is grown in near-neutral
soils, which are relatively rich in nutrients, especially
in P and Ca.
Arbuscular-mycorrhizal (AM) colonization was
present in all the cacao plantations studied. Percent
colonized root length varied between 14% and 69%. A
negative correlation was found between % AM infection in cacao roots and available soil P (p <0.05).
Spore counts varied from 38 and 1674 spores 100
g - i soil and were higher in the nursery than in the
oldest plantations (Table 3). Fifteen species of AMF
spores were observed across all the sites. Acaulospora scrobiculata Trappe was found on all sixteen sites
and Glomus constrictum Trappe was found in 73% of

The results indicate that cacao cultivation does not

reduce the diversity of AMF species in the soil after
many years of continous cultivation. In Venezuela, the
presence of the trees used to shade the cacao may help
to promote a diverse community of AME
The spore numbers recovered from cacao plantations are relatively lower than those reported for other
tropical crops like cassava (Sieverding, 1991) and coffee (Toro and Herrera, 1987). Spore counts were higher in the nursery than in older plantations as expected
because in the nurse12, cacao seedlings were kept in 3
kg plastic bags, during 6 months. These soil bags acted
as a "trap" pot culture for propagation of AME
The finding that the older plantations had the lowest spore numbers can be explained because they are
comparable with a simplified tropical forest with two
canopy layers (the shade-trees as the overstory and

319

Table 2. Soil chemical analyses of the cacao plantations studied

N

pH
(H20)

pH
(KC1)

O.M.
(%)

Niot
(%)

Pexch
(/~g g - I )

Ca2+
K+
Mg2+
(creole kg- I )

I
2
3
4
5

7.2
6.8
6.5
7.1
6.3

6.7
6.4
6.0
6.6
5.8

4.84
2.57
4.66
2.86
2.70

0.28
0.24
0.30
0.24
0.18

37
24
10
39
114

18.8
14.1
13.6
13.5
10.01

0.24
0.15
0.16
0.50
0.29

2.63
1.79
2.40
5.82
3.95

7.0

6.4

2.54

0.18

63

10.2

0.33

4.01

7.0

6.6

1.44

0.12

59

8.5

0.48

2.82

6.0

5.5

2.73

0.23

40

7.0

0.27

2.25

7.0

6.2

5.06

0.30

24

11.4

0.24

2.04

10

6.6

6.0

3.39

0.16

15

5.2

0.15

1.40

11

6.2

5.4

4.46

0.24

12

6.5

0.21

1.75

12

6.3

5.7

3.18

0.22

18

5.4

O.1l

1.37

13

7.0

6.5

4.51

0.26

18

5.5

0.14

1.65

14

6.5

5.8

2.43

0.17

12

6.3

0.15

1.86

15

7.8

7.4

4.46

0.35

24

14.3

0.16

2.98

16

6.5

5.9

9.50

0.54

14

11.3

0.35

2.80

Table3. Mean ( S.D.) spore density, richness, diversity index. Shannon Wiener index and dominance concentration
of AMF communities in cacao plantations of different ages
Plantation age

Spore density
(number 100 g-I soil)

Richness

Diversity
index

ShannonWiener
index

Dominance
Concentration

1674 4- 1216

8.67 4- 1.53

3.97 4- 0.30

0.75 4- 0.13

0.25:5::0.09

Young
(< 20 years old)
(n = 3)

448 4- 370

11.00 4- 3.00

4.33 4- 1.26

0.65 4- 0.06

0.30 4- 0.04

Mature
(20-30 years old)
(n=3)

433 + 503

8.33 4- 3.21

4.22 :t: 1.57

0.60 4- 0.25

0.42 0.23

Old
(4~70 years old)
(n = 4)

189 -4- 45

10.50 4- 3.70

4.95 4- 1.42

0.78:J:_0.05

0.22 4- 0.05

Nursery
(6months)
(n = 3)

n = number of cacao plantations considered calculating mean values.

c a c a o plants as the u n d e r s t o r y ) . A s in m a t u r e e c o s y s t e m s , A M i n f e c t i o n w a s well e s t a b l i s h e d o n c a c a o

plants. T h e i n f e c t i o n o f n e w r o o t s p r o b a b l y o c c u r r e d

Acaulospora scrobiculata w a s f o u n d in all the p l a n tations. S u r p r i s i n g l y w e did not find Scutellospora

spp., w h i c h are h i g h l y d i v e r s e in the tropics (Walker,

d i r e c t l y f r o m r o o t to r o o t in a m a n n e r m o r e r a p i d and
efficient than f r o m g e r m i n a t i n g s p o r e s (Barea et al.,

1992). S i q u e i r a et al. (1989) also r e p o r t e d that Scutellospora and Gigaspora w e r e less f r e q u e n t in c u l t i v a t e d

1991).

than in n o n - d i s t u r b e d sites.

320
Table 4. Spearman rank correlations between the calculated indexes for AMF communities, spore
populations from individual species of AMF, and edaphic properties

Available P
Index
Richness
Diversity
(sensu Whitakker)
Shannon-Wiener
Dominance
concentration
Spore populations
Acaulospora rehmii
A. scrobiculata
GIomus constrictum
G. etunicatum
Glomus sp t
Glomus sp2
Glomus sp3
Sclerocystis sinuosa

O.M.

Ntotal

Caexch

Mgech

Kexch

-0.589*
-0.599*

0.255
0.198

0.318
0.181

-0.191
-0.323

-0.067
-0.200

-0.211
-0.263

-0.446
0.425

0.473*
-0.542*

0.234
-0.280

-0.415
0.390

-0.292
0.319

-0.106
0.101

0.286
-0.157
-0.304
-0.165
0.462*
-0.599*
-0.516*
-0.347

-0.251
-0.09
0.019
0.303
-0.060
0.270
0.291
0.082

-0.007
0.094
0.046
0.358
-0.010
0.271
0.394
0.145

0.227
-0.055
-0.141
0.526*
-0.018
-0.338
0.380
-0.322

0.408
0.180
-0.033
0.066
0.481"
-0.314
-0.003
-0.115

0.151
0.114
0.303
0.086
0.590*
-0.459*
-0.122
-0.372

* Significant at p = 0.05.

The experiment with Vigna luteola shows that

cacao plants are colonized also by Sclerocystis sinuosa
and Acaulospora mellea. It is interesting to note that
although the experiment was not performed using the
roots from all the plantations studied, the three species
that were most readily trapped out with V. luteola also
were the most frequent A M species found in the fields
(Figure 1).
There are not many published data on the diversity of other A M F communities, for comparison with
cacao plantations. A n et al. (1993) encountered over
a dozen A M F species in agricultural or unmanaged
habitats. In our study, the values for species richness
varied between 3 and 14 with an average of 8.9 for all
plantations.
In relation with the diversity index, sensu Whittaker, there are no comparable data in the literature except
that of Cuenca and De Andrade (1996). Using the same
sampling procedure, they reported values of 3.9 and 4.0
for a sclerophyllous shrubland and an evergreen forest
in Venezuela, respectively. In cacao plantations, the
average value was 4.3. Thus, the diversity of A M F
spores associated with cacao is similar to that in the
natural undisturbed ecosystems in Venezuela. There
are not published Shannon-Wiener Index values for
tree crops for comparison with cacao.
Our results also indicate that the exchangeable P
in soils is correlated with a less diverse A M F commu-

nity, although more data is needed to reach a general

conclusion. The negative correlation between soil P
and colonization levels can be explained by the well
known depressive effect of P on A M infection (Bruce
et al., 1994).
The relationship between the orginic matter and
the diversity of A M F species might reflect that A M F
have a some saprophytic capacity (Hepper and Warner, 1983). The rank correlations between the individual species populations and edaphic properties revealed
some autoecological characteristics of these isolates.
For example, Glomus spl seems to be a more tolerant
isolate than Glomus sp2 and sp3 to relatively nurient
rich soils. However, it would be neccesary to have
more sampling data about them, before drawing general conclusions about their behavior.
In conclusion, the results indicate that cacao cultivation in Venezuela, maintains a diverse A M F community with well established A M colanization in roots.

Acknowledgements
The senior author gratefully acknowledges the International Foundation for Science for their support to this
project through the Grant N D/1054-2; especially Dr
Sabine Bruns who supported the development of our
laboratory and mycorrhiza research group at IVIC. We

321

.~~ t00- ]
9080-

.:,

70SO-

50-

40-

>, 3 0 ,,,

II
II
II

2o-

-- 100

I
3

'i

,, . . . . . . . .
i

H,n
i

6 7 8 9 10 tt 12 13 14 t5

Acaulosporo serobiculata Troppe

= Glomus constricturn Trappe

4 =
5=
6=
7=

8=

9=
t0=
It:
t2=
t3=
t4=
t5=

G. etuniCoturn Becket 8~ Gerdernann

Sclerocystis sinuoso Gerdernonn & Bokski
Glornus sp2 Tulasne 8~ Tulasne
Glornus spt Tulosne & Tulosne
Glornus sp3 Tulasne E~ Tulosne
Acaulosporo rehrnii SieverdinQ a Taro
G. claroideurn Schenck ~ Smith
Gigosporo sp. Gerdernann ~ Troppe
A. excovato Ingleby 8~ Walker
,~. rnorrowiae Spain a Schenck
A.rnellea Spain 8t Schenck
A.aopendicula Spain, Sierverding ~ Schenck
A. bireticulato Rothwell & Troppe

Figure 1. Frequency of AMF species in cacao plantations.

also acknowledge, the assistance of Zita De Andrade

and Gladys Escalante who helped during all the steps
of this research. Also we gratefully acknowledge the
helpful discussions with Dr Rafael Herrera and Clara
Alarc6n. We also wish to express our gratitude to the
cacao farmers who permitted us to sample their plantations. Berta Sfinchez typewrote the manuscript.

References
Allen M F and Boosalis M G 1983 Effects of two species of VA
mycorrhizal fungi on drought tolerance of winter wheat. New
Phytol. 93, 67-76.
An Z Q, Hendrix J W, Hershman D E, Ferriss R S and Henson
G T 1993 The influence of crop rotation and soil fumigation
on a mycorrhizal fungal community associated with soybean.
Mycorrhiza 3, 171-182.

Aranguren J, Escalante G and Herrera R 1982 Nitrogen cycle of

tropical perennial crops under shade trees I1. Cacao. Plant and
Soil 67, 259-269
Azizah-Chulan H and Ragu P 1986 Growth response of Theobroma cacao L. seedlings to inoculation with vesicular-arbuscular
mycorrhizal fungi. Plant and Soil 96, 279-285
Barbour M G, Burk J H and Pitts D 1987 Terrestrial Plant Ecology.
The Benjamin/Cummings Publ. Co, California, USA. 634 p.
Barea J M, Azc6n-Aguilar C, Ocampo J A and Azc6n R 1991
Moffologfa, anatomia y citologfa de las micorrizas vesiculoarbusculares, lit Fijacidn y Movilizaci6n Biol6gica de Nutrientes (Vol II). Eds. J M Barea and J Olivares. pp 14%173.
CSIC, Madrid, Spain.
Bruce A, Smith S E and Tester M 1994 The development of mycorrhizal infection in cucumber: effects of P supply on root growth,
formation of entry points and growth of infection units. New
Phytol. 127, 507-514.
Cuenca G and De Andrade Z 1996 Ecology of Glomalean populations in natural and disturbed ecosystems from La Gran Sabana,
Venezuela. In Mycorrhizas in integrated Systems from Genes to
Plant Development. Eds. C Azc6n-Aguilar and J M Barea. pp
89-92. European Commission, Luxembourg.
Cuenca G. Herrera R and Meneses E 1990 Effects of VA mycorrhiza
on the growth of cacao seedlings under nursery conditions in
Venezuela. Plant and Soil 126, 71-78.
Dodd J C, Arias I, Koomen I and Hayman D S 1990 The management
of populations of vesicutar-arbuscular mycorrhizal fungi in acidinfertile soils of a savanna ecosystem. II. The effects of pre-crops
on the spore populations of native and introduced VAM-fungi.
Plant and Soil 122, 241-247.
Ezeta F N and Santos O M 1981 Importancia de endomicorriza na
nutricao mineral do cacaueiro. R. Bras. Ci. Solo 5.22-27.
Giovannetti M and Mosse B 1980 An evaluation of techniques for
measuring vesicular-arbuscular mycorrhizal infection in roots.
New Phytol. 84,489-500.
Harley J L and Smith S S 1983 Mycorrhizal Symbiosis. Academic
Press. London, UK.
Hepper C and Warner A 1983 Role of organic matter in growth
of a vesicular-arbuscular mycorrhizal fungus in soil. Trans. Br.
Mycol. Soc. 81, 155-156.
Jackson M L 1976 Anfilisis Qufmico del Suelo. Edic Omega SA,
Barcelona, Spain.
Jeffries P and Barea J M 1994 Biogeochemical cycling and arbuscular mycorrhizas in the sustainability of plant-soil systems. In
Impact of Arbuscular Mycorrhizas on Sustainable Agriculture
and Natural Ecosystems. Eds. S Gianinazzi and H Scdhepp. Adv.
Life Sci. pp 101-115. Birkh~iuser Verlag, Basel, Switzerland.
Johnson N C, Pfleger F L, Crookston R K, Simmons S R and
Copeland PJ 1991 Vesicular-arbuscular mycorrhizas respond to
corn and soybean cropping history. New Phytol. 117, 657-663.
Johnson N C, Tihnan D and Wedin D 1992 Plant and soil controls
on mycorrhizal fungal communities. Ecol. 73, 2034-2042.
Laycock D H 1945 Preliminary investigations into the function of
the endotrophic mycorrhiza o f Theobroma cacao L. Trop. Agric.
XXII, 77-80.
Murphy J and Riley J P 1962 A modified single solution method for
the determination of phosphate in natural waters. Anal. Chim.
Acta 27, 31-36.
Olsen S R, Cole C V, Watanabe F S and Dean L A 1954 Estimiation of available phosphorus in soils by extraction with sodimn
bicarbonate. US Dept. Agric. Cir. 939.
Phillps J M and Hayman D S 1970 Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycor-

322
rhizal fungi for rapid assessment of infection. Trans. Br. Mycol.
Soc. 55,158-161.
Siegel S 1978 Estad~stica no param6trica aplicada alas ciencias de
la conducta. Editorial Trillas, Mexico City, Mexico. 346 p.
Sieverding E 1991 Vesicular-Arbuscular Mycorrhiza Management
in Tropical Agrosystems. GTZ, Eschborn, Germany.
Siqueira J O, Colozzi-Filho A and Oliveira E 1989 Ocurrencia de
micorrizas ves[culo-arbusculares em agro e ecossistemas naturais do estado de Minas Gerais. Pesqui. Agropecu. Bras. 24,
1499-1506.
Toro M and Herrera R 1987 Existence of mycorrhizal spores in two
different coffee plantations. In Mycorrhizae in the next decade.
Eds. D M Sylvia, L L Hung and J H Graham. p. 60 Proc. 7th.
NACOM Gainsville, Florida, USA.

Walker C 1992 Systematics and taxonomy of the arbuscular endomycorrhizal fungi (Glomales)- a possible way forward. Agronomie
12, 887-897.
Whittaker R H 1975 Communities and Ecosystems, Second ed.
McMillan Publishing Co, Collier MacMillan Publ., New York,
London, UK. 385 p.
Section editor: J H Graham