J. Euk M i c r o b i d , 44(2). 1997 pp. 122-126 6 1997 by the Society of Protozoologists

Species Richness and Abundance of Naked Amebae in the Rhizoplane of the Desert Plant Escontriu chiotilla (Cactaceae)

SALVADOR RODRiGUEZ-ZARAGOZAI and SOLEDAD GARCiA Luboratop of Microbial Ecology, Unidad de Investigacidn Interdisciplinaria en Ciencias de la Sulud y la Educacidn,

Universidad Nucional Autdnoma de MPxico, Campus Iztucah, Apartado Postal 16-491 Azcupotzalco, Distrito Federal. Cbdigo Postal 0201 I , Mkxico

ABSTRACT. The species richness and quantity of naked amebae were determined in the bulk soil and rhizoplane of the desert plant Escontria chiotilla in the Valley of Tehuacan, Mexico. Samples from bulk soil were taken at 10-cm and 30-cm depths in April, May and July, 1993, and from roots and soil at a 10-cm depth in June and July, 1994. Quantity of amebae obtained by Most Probable Number method increased in the rhizoplane by two orders of magnitude after rains. Likewise, the countable population of amebae doubled in numbers at both the 10- and 30-cm depths after rains. We isolated 163 strains from both root and soil environments, which were grouped into 40 bactivorous and/or generalist species belonging to 19 genera. Species richness showed no clear dominance of a particular genus in either bulk soil or root. Acanthamoeba (groups I1 and 111, Pussard & Pons) and Vahlkampfiu accounted for 12.5% and 15% of the total number of species, respectively. However, greater species richness was found in bulk soil than on root surfaces. We concluded that the diversity of naked amebae, taken as numbers of individuals (or as biomass) of each species and its evenness, is still needed to assess the ecological roles of Acanthamoeba and Vahlkampfia in the soil environment.

Supplementary key words. Acanthamoeba, bactivorous free-living amebae, soil protozoa, Vahlkampfiu.

ICROBIOLOGICAL studies of the rhizosphere and rhi- M zoplane have previously focused on bacteria and my- corhizas. Little or no attention has been directed to naked ame- bae or to interactions among them and with fungi, bacteria and algae. Even when free-living amebae (FLA) are not the only predators in soils, they have been recognized as the main con- trollers of bacterial population growth due to their fast response to bacterial increases [5-7, 241. While nematodes are also very important bacterial grazers [20], they tend to feed more readily on amebae than on bacteria [7].

As a group, FLA may feed on bacteria, fungi, algae and other protozoa, including other amebae. However, studies on naked amebae are focused mostly on bacterial grazers [25]. Singh [3 I] pointed out that nonpigmented bacteria can support the growth of amebae, while some species are only used in emergency cases and pigmented bacteria are usually unedible, due to the presence of compounds toxic to amebae. Prior to Singh, Svert- zova [34] had reported that several species of amebae showed prey preference for pigmented or spore-forming bacteria, but no other report has supported this fact to date. Weekers et al. [36] have reported that Hartmannella vermz~ormis may feed temporarily on pigmented bacteria, such as Chromatium vinos- um and Serratia marcescens, giving more support to Singh's statements.

Studies concerning bactivorous amebae in the rhizosphere have reported the change of density of amebae as a response to changes in bacterial density 15-71, without mentioning the variety of species present in their samples. However, four gen- era of naked amebae are frequently reported in studies on soil protozoa, i.e. Acanthamoeba, Hartmannella, Naegleria and Vahlkamp3a [ 8 , 14 ,30, 31, 33, 341. This may be explained by the lack of emphasis placed on FLA during the first half of the century and by the increased interest in pathogenic amebae after the discovery of primary amoebic meningoencephalitis [ 191. It is unlikely that these genera are the only ones present in soils. Their ecological importance may have been misunderstood due to lack of information about their diversity, abundance and prey preferences. On the other hand, soil microbial food webs have been recognized very recently as an important factor to be con- sidered when studying plant growth [ 151, plant productivity and interactions with soil-borne plant pathogens [2].

The objectives of this study were to determine the total num- ber of amebae in and around the rhizoplane of the desert cactus

I To whom correspondence should be addressed. Telephone: 52-5- 560- 13 10, Fax: 52-5-565- 1009, Email: srodrige@mailer.main.conacyt.mx

Escontria chiotilla (Weber) Rose, 1906, during both dry and wet seasons, and to describe the taxonomic distribution of na- ked amebae in this environment.

MATERIAL AND METHODS

Escontria chiotilla was chosen for this study to continue a research program on the ecophysiology of this plant. This cac- tus is of economic importance for the Tehuacan human popu- lation because its fruits are used for manufacturing candies, as a food source [3], and for ice cream and liquor. However, fruits are collected by individuals without any regulation. Lambs and goats feed on young plants, and sometimes mature ones are cut for fodder when the weather becomes dry.

Description and localization of plant population site. Our E. chiotilla samples were collected between 18" 10' 8" N lati- tude and 97" 10' to 97" 15' E longitude in the northern part of the valley of Tehuacan (Coxcatlan county, Puebla, Mexico), which is at an altitude of 1,220 m above sea level and which has an annual mean temperature of 27" C [35]. The climate has been classified as BSh (131, indicating an annual mean precip- itation in the range of 400-575 mm over 90 days from May to October. The soil has two horizons (A and C) and three types of texture, i.e. loamy, sandy-loam and sandy-clay. Common pH ranges from 7.4 to 8.3 because calcium carbonate is very abun- dant. The region is primarily flat with small elevations of light slopes and some hills and depressions. Vegetation consists of grasses, spiny forests and xerophytic shrubs [27].

Long et al. [ 181 proposed that this locality is part of the area where corn (Zea mays L.) was domesticated and cultivated [12, 181. The human population in the Coxcatlan area dates back to 6,000 years before present, when the early plant domestication took place. Thus, there is the possibility that our study site in the Tehuacan valley could have been used for cultivation a long time ago, but this has not been the case for over 500 years.

Collection and processing of samples. Soil and root sam- ples were collected from three individuals chosen at random from a population of E. chiotilla. Soil and root samples were taken at depths of 10 and 30 cm, approximately 40-60 cm away from the stems, in areas where grasses and other cacti were absent. Samples were taken in May, April and July, 1993, but roots and soil samples were taken only at the 10-cm depth dur- ing June and July, 1994. Soil was air dried and then sieved through a 2-mm mesh.

One gram of soil was mixed with 2 ml sterile distilled water, vortexed for 20-40 s four times, and then left undisturbed for 10- 15 min for sand sedimentation, the supernatant was then used

122

RODR~GUEZ-ZARAGOZA & GARCIA-ABUNDANCE OF FLA IN THE RHIZOPLANE 123

Table 1. Quantity of free-living amebae in the rhizoplane. The num- ber of free-living amebae per gram of root increases one to two orders of magnitude from the dry month (June) to the early rainy season (July). Data were obtained from soil surrounding root surfaces (the rhizoplane) of three individuals of E. chiotilla.

~ ~~

Quantity of amebae per gram of root

Season Individual 1 Individual 2 Individual 3

Dry (June 94) 127.2 104.1 94.5 Wet (July 94) 5,850.0 18,333.6 5,687.7

immediately for estimation of the most probable number (MPN), as described by Singh [30] and modified by Darbyshire et al., [lo]. Root samples were kept in plastic bags following collection. Then, we weighed 1 g of roots and washed them thoroughly with 3 ml sterile distilled water. These washes were used for estimation of MPN in the same way as the soil samples.

Estimation of MPN consisted of 15 twofold dilutions with eight replicates each, as follows 1 5 ; 1:lO; 1:20; 1:40; 1:80; 1: 160; 1:320; 1: 640; 1: 1,280; 1: 2,560; 1: 5,120; 1: 10,240; 1: 20,480; 1: 40,960 and 1:81,920. The dilution mixtures were prepared with Neff’s saline solution (NaCl 0.12 g/l, MgS0,.7H20 0.004 g/l, CaC1,.2H,O 0.004 g/l, Na,HPO, 0.142 g/l and KH,PO, 0.136 g/l) [22] plus heat killed Enferobacter aerogenes and the corresponding proportion of sample as di- rected by the microtiter method [lo]. Positive growth was reg- istered after incubation at 30” C for 10 days. The total numbers were calculated by Thomas’ formula (described in [ l]), and the numbers reported here are the geometric means. Samples from greater dilutions that exhibited growth were observed under phase contrast microscope for determination of genera.

The taxonomic analysis was done by direct inoculation of samples in nonnutritive agar plates containing heat-killed bac- teria. After cultivation for 7 days, each clone was identified by morphology with the aid of a phase contrast microscope and Page’s key for freshwater and soil amoeba 1221. The genera of Vahlkampfidae were diagnosed by a flagellation test, as de- scribed by Page [22]. Heterolobosea strains that fail to flagellate after encubation at 35” C during 48 h in sterilized distilled water were assigned to the genus Vahlkampjia, and the amoebofla-

Table 2. Changes in number of free-living amebae in bulk soil at 10- and 30-cm depth.

Number of amebae per gram of soil sample

Samples taken at 10 cm Samples taken at 30 cm

Month Soil 1’ Soil 2 Soil 3b Soil 1 Soil 2 Soil 3

April 93 2,351 1,146 498 302 266 150 May 93 Missing 272 124 303 229 1,359

July 93 3,352 2,825 1,237 880 807 451 June 94 Od 85 126 Not investigated‘

July 94 4,353 4,206 1,117 Not investigated

valuec

a Sandy-loam. I, Loam. =The sample was processed, but was disposed by accident before

information was registered. We got no growth in any well. This gave us zero amebae present in

the sample. However, we are unable to state no amebae were there due to the error introduced by the counting method.

eThe samples from 30 cm were not processed before 10 days of sampling.

Table 3. Presence of species in soil and rhizoplane of E. chioritla.

Species Roots 1 0 c m 3 0 c m ~ -~

Acanthamoeba sp‘ + + + A. quina + + + A . polyphaga + + + A . lenticulata + A. rhizodes + Adelphumoeba galeacystis - Cashia sp. -

Cochliopodium actinophorum -

Cochliopodium bilimbosum - Commandonia operculata + Dermamoeba minor + Echinamoeba sp. i t + E. exundans t + E. silvestris + t + Filamoeba sp. +

+ + F. nolandi -

Glaeseria mira + + + Hartmannella sp. t i + H. cantabrigiensis + + H . vermiformis t + + Hyalodiscus sp. - Mayorella sp. - Naegleria sp. -

N. gruberi t + Plaryamoeba sp. + + + P. placida + Jr

P. stenopodia + t Protacanrhamoeba sp. + + Vahlhmfia sp. + + +

+ V. aberdonica - V. avara t + V. froschi + + V. magna V. ustiana i + Vannella sp. + V. mira t V. platypodia + Vexilifera telma - Tetramirus sp. -

- - - -

- + + t +

- - + C. limacoides -

- -

- -

- -

-

- -

-

- + + + +

-

-

- - -

- - -

+ - -

- - -

- -

- - - +

+ -

‘The abbreviation sp. denotes those strains that did not match with the description of any species provided by the key, and means “an unidentified species.”

gellates were assigned into either Naegleriu, Tetrumitus, Par- atetramitus or Adelphamoeba based on the number of flagella, morphology of the flagellate itself, and excystment pores. Strains of Acanthamoeba were determined at the group level by cyst morphology as proposed by Pussard & Pons [23], and were assigned into a species based on morphological data.

RESULTS The quantity of amebae living on roots surfaces changed

markedly between seasons, increasing more than two orders of magnitude after rains (Table 1). A similar change occurred in bulk soil, where differences between the dry and wet seasons are also of two to three orders of magnitude at the 10-cm depth (Table 2). There were also significant changes at 30-cm depth, allowing for a doubling of the population. Amebae were always more numerous in the upper layer of soil, which is in accor- dance with the general assumption that protozoa are more abun- dant in the first 15 cm of soil. This is also true for the rhizo- sphere of these plants, because Escontria roots tend to spread horizontally rather than vertically into the desert soil [3].

The genus Echinamoeba (7-15 pm) was almost always ob- served, even in up to the highest dilutions during the rainy

124 J. EUK. MICROBIOL., VOL. 44, NO. 2, MARCH-APRIL 1997

:ies Richness

r i ronments

L OJ

u. - .-

Fig. 1. Species richness of FLA in bulk soil and on the rhizoplane of E. chiotiffa. Figure shows the proportion of species by genus in each microenvironment. Acanthamoeba, Echinamoeba, Hartmannella, Platyamoeba, Vahlkampfia and Vannella represent 78% of the variety in the rhizoplane. However, they represent only 55% in bulk soil as a consequence of having more genera represented in soil. The proportion of species richness for each genus was obtained taking in account the species present on roots or bulk soil. Meanwhile the column “Both Environments” shows only those species that were present in both microenvironments.

season. Meanwhile, cysts of Acanthamoeba and Vahlkampjia (18-35 p,m) were present only in the lowest dilutions. A total of 163 strains were cloned from both microenvironments, bulk soil and roots. Morphological identification of these strains showed that 40 species and 19 genera were represented in our samples (Table 3). Vannella and Mayorella are noncyst-forming amebae and have not been reported as endosymbionts, but somehow, they are able to survive in the desert soil.

Vahlkampfia and Acanthamoeba were the best represented in both bulk soil and roots. However, we found no clear domi- nance of a particular genus. The representation of these genera was the same when roots and bulk soil were taken separately, but Acanthamoeba represented 18% and Vahlkampjia 14.8% of the species richness on the surface of roots. The opposite oc- curred in the bulk soil samples where Acanthamoeba and VahlkampJia accounted for 9.4% and 18.7% of the species rich- ness respectively (Fig. 1). Species richness was higher in bulk soil at thelO-cm depth than in root surfaces or in bulk soil at 30 cm (Table 3). Almost 50% of the species were present in both root and bulk soil samples, 17.5% were found only on root samples, and 32.5% were found only in bulk soil samples.

DISCUSSION

Water availability is recognized as the main limiting factor in desert environments and plant productivity is actually syn- chronized with the rainy season, which may lead one to think that microbial-protozoal processes are determined by this syn- chronization too. However, some microbial soil processes may be nonsynchronous with rainfall, as may be the case for nitro- gen mineralization as hypothesized for the Negev Desert in Is- rael [32]. Due to scarcity of the rainfall events, desert soil mi- crobiota must be adapted to activate quickly when water be- comes available, and then reduce their activity gradually as wa- ter becomes more scarce. This ability is shared by other desert organisms, including plants. The increase in the number of ame- bae in the rhizoplane may be a consequence of the physiolog- ical activation of plants, which produces root exudates and en- hance bacterial growth at the beginning of the rainy season. Plants become active in order to produce and store all the ma- terials needed for the next flowering time, which begins late in the dry season. In the same way, the scarcity of amebae may be explained by low bacterial activity, due to the dormant stage

RODRIGUEZ-ZARAGOZA & GARCfA-ABUNDANCE OF FLA IN THE RHIZOPLANE 125

of the roots at that time. It has been established that most pro- tozoa can feed actively under a very narrow range of soil water content [37], which would eliminate their influence in this en- vironment during the driest periods. The rhizosphere environ- ment largely differs from that of the bulk soil because it de- pends on the presence of bacteria, which feed on plant exudates, to support the growth of protozoa. On the other hand, activity at different soil humidities has been investigated only for cili- ates and flagellates 19, 11, 371. Amebae do not need thick water films for swimming and this would allow them to be active at lower levels of soil humidity. More research is needed on the relationship between soil-water levels and ameba1 activity. However, changes in the abundance of amebae during the dry season may also be related to urine provided occasionally by mammals that forage on the herbaceous plants around the cacti. This phenomenon is being recognized as an important link among the herbivory aboveground and the microbial processes belowground in savanna grasslands 1281. This may be the case with lambs and goats that forage in our zone of study. These events, along with dew and other wetting processes, must be kept in mind for further studies on microbial predator-prey dy- namics under normal soil environments. The addition of urine itself causes large increases of bacteria, in numbers dispropor- tionate to the amount of water added to soil [28]. The excretions of mammals also add organic matter into a system that is or- ganic carbon-limited [28] , which may enhance microbial pro- ductivity even more.

The numbers of organisms found in this survey are consistent with previous findings that protozoa are generally more abun- dant in the first 15 cm of soil, despite the fact that the strongest changes of temperature in the desert soil occur in this layer. The population of protozoa decreases below the first 15 cm of soil and may be concentrated in the water table where they flourish again if sufficient food is available [21].

The genera found in our samples represent only the bacti- vorous amebae and those species that can survive by tempo- rarily feeding on bacteria as an alternative source of food. While our data do not indicate the quantities of resources uti- lized by each genus, the variety of genera detected in our sam- ples indicates that a complex amebae community shares the resources provided by the E. chiotilla rhizosphere. Further stud- ies on the diversity of naked amebae in undisturbed environ- ments are needed in order to know the ecological value, the number of individuals, the biological activity, and the distri- bution of species inhabiting different soil microenvironments.

Acanthamoeba constitutes a higher proportion of the species richness in disturbed environments than in undisturbed areas. For example, this genus may account for up to 45% of the amebae recovered from the urban air [26], but only accounted for 12.5% of the species richness in the present study. This difference is explained by the ability of several species of Acan- thamoeba to tolerate hydrogen sulphide [17] and anoxia 1161 as trophozoite, and to the cyst stage, which is very resistant to many adverse factors including chlorine [19], but this genus may be unable to dominate in the same way in undisturbed environments due to the lack of factors that kill other species. It is also noteworthy that only Acanthamoeba from groups I1 and I11 were present in our samples.

The fact that bulk soil bore more species of amoeba than did root surfaces leads us to think that the first 15 cm of the soil is the main reservoir of these protozoa. The number of genera represented in our samples was high and with few species rep- resenting each, as expected in an undisturbed environment.

The variety of species may be explained as a normal response of the group to a higher quantity of prey present under common environmental conditions. This could be true, as long as the soil

where this cactus population grows has not been used for ag- riculture. However, the variety of naked amebae may be greater than that obtained in this study, because the 40 species identi- fied could grow feeding on E. aerogenes at least for short pe- riods of time. There could be more species that cannot be ob- served unless the appropriate food is provided, as pointed out in Chakraborty & Old [4] for mycophagous amoebae. Carniv- orous, fungivorous and algivorous species are still unaccounted for due to the mode of cultivation used here as it is effective only for bactivorous amebae.

We need to increase studies of diversity, abundance, impor- tance, rate of biomass turnover, nitrogen mineralization rates, and predator-prey interactions of naked amebae, to integrate this knowledge into the ecological dynamics of soil microin- vertebrates, nematodes, and oligochaeta. This will allow us to draw a better picture of the importance of microbial cycling of matter, energy turnover, and its importance for conservation and recovering of soil resources. In the long term, it would lead to improved practices of soil management.

ACKNOWLEDGMENTS We wish to thank Antonia Trujillo and Manuel Mandujano

for their invaluable help during field sample collection, and to the anonymous reviewers for their very useful comments.

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75: 892-904.

Received 1-9-95, 4-25-96, 9-25-96; accepted I 1-13-96