Plant and Soil 183:315-322, 1996. 315 @ 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 symbi- otic 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 poten- tial 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 tra- ditional 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 1000- 2500 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 iso- lates 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 com- munities 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.

Materials and methods

Study site

Sixteen cacao plantations and one nursery from north- ern 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 (Lau- raceae) and Artocarpus altilis (Moraceae) (Table 1). Farmers also use several species of Musa as a tempo- rary 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

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.

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). Per- centage infection was assessed by the gridline method of Giovannetti and Mosse (1980). A minimum of 100 intercepts were scored for each sample.

Arbuscular-mycorrhizal fungi communities infield soils

To study the AMF communities, three composite sam- ples 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 sam- pled, but the spores were isolated from several differ- ent seedling cohorts. Soil samples were maintained in closed plastic bags at 4 C until processing, no lat- er 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 centrifu- gation (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. Morphologi- cally distinct spores were checked again using a com- pound microscope. From each different morphological type, a permanent slide was prepared using polyvinyl- lactoglycerol 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 (Scle- rocystis 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 domi- nance concentration for each sampled site according to the following equations:

Richness: R = Number of species found in a sample.

Diversity: D : S / logN

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

317

N Site Aprox. age Shaded by Management

I Boca de Caucagua. 30 years

Barlovento, Miranda State 2 Boca de Caucagua, 30 years

Barlovento, Miranda Stale

3 Tapipa Grande, >70 years

Barlovento, Miranda State 4 a Ocumare de la Costa, 2 years

Hacienda Monasterio, Aragua State

5 a Ocumare de la Costa. > 20 years

Hacienda Monasterio, Aragua State

6 ~' Ocumare de la Costa, > 30 years Hacienda Monasterio.

Aragua State

7 La Esmeralda. Ocumare de > 70 years la Costa, Aragua State

8 a Santa Cruz de la Vega, 5 years

Aragua State

9 Ri'o Seco, Yaguaraparo, 40-50

Sucre State years 10 a Rio Seco, Yaguaraparo, > 30 years

Sucre State

11 Los Palmares, 3M years Yaguaraparo, Sucre State

12 Rio Seco, Yaguaraparo > 30 years

Sucre State 13 Rio Seen, Yaguaraparo, > 30 years

Sucre State 14 Ri'o Seco Yaguaraparo. 4-5 years

Sucre State

15 Yaguaraparo, Sucre State 6 years

16 Loma Maffn, San Jos6. 30-40

Sucre State years Nut- Panaquire. Miranda Stale 0-6 months sery

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

Erythrina poeppigiana. Musa sp.

Eiythrina poeppigiana. Musa sp.

Tabebuia pentaphylla, Ervthrina poeppigiana Etythrina poeppigiana

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

Cupravit b and prunning

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.

Where S = total number o f spec ies in the sample , and

N = total number o f spores in the sample (Whit taker ,

1975).

Where C is the index number , s is the total number

o f spec ies in the sample , and p is the propor t ion o f all

spores in the sample that be long to spec ies i (Barbour et al., 1987).

Dominance Concent ra t ion :

(S impson 's index)

s

C = y~(p i ) 2

i=l

s

Shannon-Wiener D ivers i ty Index H I ---- - ~ pi In pi

i= l

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 colo- nizing cacao roots, fine cacao roots from the five plan- tations 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 individ- uals in each plantation were carefully washed with tap water and sonicated for 5 min to eliminate soil parti- cles. 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 steril- ized soil collected from Ocumare plantations. A treat- ment 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) trans- formed 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 individu- al species and edaphic characteristics were calculated according to Siegel (1978).

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 infec- tion in cacao roots and available soil P (p

Table 2. Soil chemical analyses of the cacao plantations studied

N pH pH O.M. Niot Pexch Ca2+ K + Mg 2+ (H20) (KC1) (%) (%) (/~g g- I ) (creole kg- I )

I 7.2 6.7 4.84 0.28 37 18.8 0.24 2.63 2 6.8 6.4 2.57 0.24 24 14.1 0.15 1.79

3 6.5 6.0 4.66 0.30 10 13.6 0.16 2.40

4 7.1 6.6 2.86 0.24 39 13.5 0.50 5.82 5 6.3 5.8 2.70 0.18 114 10.01 0.29 3.95 6 7.0 6.4 2.54 0.18 63 10.2 0.33 4.01

7 7.0 6.6 1.44 0.12 59 8.5 0.48 2.82

8 6.0 5.5 2.73 0.23 40 7.0 0.27 2.25

9 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

Table 3. Mean ( S.D.) spore density, richness, diversity index. Shannon Wiener index and dominance concentration of AMF communities in cacao plantations of different ages

319

Plantation age Spore density Richness Diversity Shannon- Dominance (number 100 g-I soil) index Wiener Concentration

index

Nursery

(6months)

(n = 3)

Young (< 20 years old)

(n = 3)

Mature

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

Old (4~70 years old)

(n = 4)

1674 4- 1216 8.67 4- 1.53 3.97 4- 0.30 0.75 4- 0.13 0.25:5:: 0.09

448 4- 370 11.00 4- 3.00 4.33 4- 1.26 0.65 4- 0.06 0.30 4- 0.04

433 + 503 8.33 4- 3.21 4.22 :t: 1.57 0.60 4- 0.25 0.42 0.23

189 -4- 45 10.50 4- 3.70 4.95 4- 1.42 0.78 :J:_ 0.05 0.22 4- 0.05

n = number of cacao plantations considered calculating mean values.

cacao plants as the understory). As in mature ecosys-

tems, AM infection was well establ ished on cacao

plants. The infection of new roots probably occurred

directly from root to root in a manner more rapid and

efficient than from germinat ing spores (Barea et al.,

1991).

Acaulospora scrobiculata was found in all the plan- tations. Surprisingly we did not find Scutellospora spp., which are highly diverse in the tropics (Walker,

1992). Siqueira et al. (1989) also reported that Scutel- lospora and Gigaspora were less frequent in cultivated than in non-disturbed 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 O.M. Ntotal Caexch Mgech Kexch

Index Richness -0.589* 0.255 0.318 -0.191 -0 .067 -0.211 Diversity -0.599* 0.198 0.181 -0.323 -0 .200 -0.263 (sensu Whitakker) Shannon-Wiener -0.446 0.473* 0.234 -0.415 -0 .292 -0.106 Dominance 0.425 -0.542* -0.280 0.390 0.319 0.101 concentration

Spore populations Acaulospora rehmii 0.286 -0.251 -0.007 0.227 0.408 0.151 A. scrobiculata -0.157 -0.09 0.094 -0.055 0.180 0.114 GIomus constrictum -0.304 0.019 0.046 -0.141 -0.033 0.303 G. etunicatum -0.165 0.303 0.358 0.526* 0.066 0.086 Glomus sp t 0.462* -0.060 -0.010 -0.018 0.481" 0.590* Glomus sp 2 -0.599* 0.270 0.271 -0.338 -0 .314 -0.459* Glomus sp 3 -0.516* 0.291 0.394 0 .380 -0.003 -0.122 Sclerocystis sinuosa -0.347 0.082 0.145 -0.322 -0 .115 -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 AM species found in the fields (Figure 1).

There are not many published data on the diver- sity of other AMF communities, for comparison with cacao plantations. An et al. (1993) encountered over a dozen AMF 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 Whittak- er, 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 AMF 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 AMF 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 AM infection (Bruce et al., 1994).

The relationship between the orginic matter and the diversity of AMF species might reflect that AMF have a some saprophytic capacity (Hepper and Warn- er, 1983). The rank correlations between the individu- al 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 gen- eral conclusions about their behavior.

In conclusion, the results indicate that cacao cul- tivation in Venezuela, maintains a diverse AMF com- munity with well established AM colanization in roots.

Acknowledgements

The senior author gratefully acknowledges the Interna- tional 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

.~ ~ ~ t00- ] 90-

80-

.:, 70 -

SO-

50-

,,, 40- 1 >, 30- 2o-

-- 10- 0 , i , '

2 3 4 5 I

= 3 4 = 5= 6= 7= 8= 9=

t0= I t : t2= t3= t4= t5=

II II II

,, . . . . . . . . H ,n i i i i i i i i

6 7 8 9 10 tt 12 13 14 t5 Acaulosporo serobiculata Troppe Glomus constricturn Trappe 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 plan- tations. Berta Sfinchez typewrote the manuscript.

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Section editor: J H Graham