Folia Microbiol. 50 (5), 421–425 (2005)
http://www.biomed.cas.cz/mbu/folia/
Variability of Non-Mutualistic Filamentous Fungi Associated with Atta sexdens rubropilosa Nests A. RODRIGUESa, F.C. PAGNOCCAa, M. BACCI Jr.a, M.J.A. HEBLINGa, O.C. BUENOa, L.H. PFENNINGb aCentro de Estudos de Insetos Sociais, UNESP, 13506-900 Rio Claro, São Paulo, Brazil bDepartamento de Fitopatologia, Universidade Federal de Lavras, 37200-000 Lavras, Minas Gerais, Brazil
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[email protected] Received 9 November 2004 Revised version 18 July 2005
ABSTRACT. A survey of the filamentous fungi other than the symbiotic one found in association with Atta sexdens rubropilosa colonies was carried out. Different fungal species (27 taxa) were isolated a few days after treating the workers with toxic baits (sulfluramid; Mirex-S®), from 40 laboratory and 20 field nests. Syncephalastrum racemosum (54 %) and Escovopsis weberi (21 %), Trichoderma harzianum (38 %) and Fusarium oxysporum (23 %) were the prevalent species in laboratory and field nests, respectively. Acremonium kiliense, Acremonium strictum, E. weberi, F. oxysporum, Fusarium solani, Moniliella suaveolens and T. harzianum were found in both nests’ groups. We revealed that many filamentous fungi can co-exist in a dormant state inside the nests of these insects and some of them appear to be tightly associated with this environment.
The well-known leaf-cutting ants of the genera Atta and Acromyrmex (Hymenoptera: tribe Attini) live in a symbiotic relationship with the fungus Leucoagaricus gongylophorus (Silva-Pinhati et al. 2004). These specialized ants are unique since they have the ability to cultivate and feed on their partners. Because huge quantities of fresh plant material are cut to maintain the symbiotic fungus, some authors have raised these ants to a pest’s status, mainly in South American countries, where major losses can occur in cultivated areas (Hölldobler and Wilson 1990). In Brazil, the control of these insects has been done with toxic baits containing sulfluramid. In addition to these two organisms involved in the symbiosis, it is known that a pool of microorganisms can be found in association with the fungus garden of these ants, such as bacteria, yeasts and filamentous fungi (Craven et al. 1970; Fisher et al. 1996; Pagnocca et al. 1996; Carreiro et al. 1997). Inter-specific relationships among some members of this microbiota were observed and for some of these microorganisms it is thought that they could help the symbiotic fungus in the degradation of the plant substrate (Bacci et al. 1995; Carreiro 2000; Carreiro et al. 2002) or to protect the ants from infections by entomopathogenic fungi (Santos et al. 2004). Recent findings have shown that the ants’ nests are, at least in relation to the microbial community, less known than previously thought. Thus, two new yeast species were recently described to occur in this environment – Cryptoccocus haglerorum (Middelhoven et al. 2003) and Sympodiomyces attinorum (Carreiro et al. 2004). In an exciting discovery, Currie et al. (1999a) described a third partner in this relationship, an actinomycete living on the worker’s cuticle. It was found that these bacteria produce unidentified substances that are specifically targeted to the suppression of growth of Escovopsis spp. The latter are ascomycete anamorphs, parasites of the fungus garden, found in association with the entire phylogenetic distribution of the Attini tribe (Currie et al. 1999b, 2003a). Escovopsis is a genus with striking morphological characteristics with only two species described, E. weberi (Muchovej and Della Lucia 1990) and E. aspergilloides (Seifert et al. 1995). Infections with this parasite can be persistent and can lead to losses of the garden biomass (Currie 2001) and, occasionally, it can overwhelm the fungus garden even in the presence of the workers. Therefore, evidence shows that Escovopsis sp. is the major fungal contaminant inside the nests of the derived attines, such as the leaf-cutting ants (Currie et al. 1999b). Beside Currie’s research, the research of nonmutualistic filamentous fungi in colonies of leaf-cutting ants has been overlooked and only a few studies were made in this respect (Möller 1893; Weber 1966; Bass and Cherrett 1994; Fisher et al. 1996). No study is available concerning the prevalence and species diversity of this group of microorganisms in the nests. Therefore, the aim of the present study was to screen filamentous fungi in the field and laboratory nests of the leaf-cutter ant Atta sexdens rubropilosa. In order to
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facilitate the growth of contaminants, the number of worker ants of the nests was intentionally reduced using toxic baits.
MATERIALS AND METHODS Nest collection and maintenance. In March 2002, 60 young colonies (age ≈5 months) of Atta sexdens rubropilosa living in the soil of an orange plantation in the vicinity of the Corumbataí City (São Paulo, Brazil; S 22°17′22″; W 47°39′23″') were collected. In laboratory the nests were maintained in plastic boxes (≈1 L) and supplied with Eucalyptus sp. leaves and oat flakes for 5 months prior to the beginning of the experiments. Isolation and identification of fungi. The laboratory nests (n = 60) were grouped randomly as follows: (i) 10 continued receiving the Eucalyptus sp. leaves, (ii) 10 received 5 g of baits without the active principle and (iii) 40 nests received 5 g of toxic baits containing 0.3 % of sulfluramid (Mirex-S®, commercial bait). For comparison, 20 young nests in the same area received ≈10 g Mirex baits. The baits were dropped close to the entrance of these nests and after a period of 6–12 d the nests were carefully opened and analyzed. In both approaches, when fungi were detected (macroscopically or using a stereomicroscope) they were direct plated on malt agar (2 %, W/V) with 150 µg/L chloramphenicol (Sigma) (Gams et al. 1998). The plates were incubated in the dark for 3–7 d at 25 ºC. Microfungi from laboratory nests were isolated from fungus gardens, waste deposits and fragments of baits, whereas fungi from field nests were isolated only from the fungus garden. Isolates were identified with available keys published by Domsch et al. (1980), Samson et al. (1995) and some other specific monographs. Cultures were preserved at Centro de Estudos de Insetos Sociais on agar slants and in vials with distilled water (Smith and Onions 1983).
RESULTS AND DISCUSSION Some filamentous fungi can develop in the fungus garden of the leaf-cutting ants when the number of workers is reduced or when they are removed, showing how important the worker ants are for the microbial control of the colonies (Möller 1893; Weber 1966; Weber 1972; Bass and Cherrett 1994; Ortiz et al. 1999); we obtained a similar result. All the 40 laboratory and the 20 field nests were considered extinct after treatment with sulfluramid and in 28 and 13 of them, respectively, several alien fungi were detected a few days after the treatment. A total of 74 strains were isolated from the former group and 23 from the latter one. In the laboratory nests, most of the isolates were growing in the fungus garden but sometimes they occurred in the waste deposit and on bait fragments as well (Table I). Sometimes more than one fungus species was isolated from the same nest. Twenty-six taxa and one sterile mycelium were identified, most of them being anamorphic ascomycetes; a few representatives of Mucorales were also found. Half of the species found in the fungus garden of the laboratory nests were also recorded in the waste deposit (Table I), showing that these microorganisms are distributed in different parts of the nest probably during the ant movement. Fungus gardens contain many nutrients derived from the metabolic activity of the symbiotic fungus towards plant cell polysaccharides (Silva et al. 2003). Most of these nutrients are simple sugars, which can be assimilated by microorganisms, including fungi, and probably this is what happens when they get out of the control of the workers in this environment. Most of the fungi were found in low numbers; a few of them also occurred at high rates. In laboratory colonies, Syncephalastrum racemosum (54 %), E. weberi (21 %), and Fusarium solani (18 %) were the predominant species. S. racemosum and E. weberi were observed growing only in the fungus garden and F. solani in the waste deposit and also in the fungus garden. On the other hand, Trichoderma harzianum (38 %), Fusarium oxysporum (23 %), and E. weberi (15 %) were the prevalent species found in the field nests. In addition, the species Acremonium strictum, Acremonium kiliense, E. weberi, F. oxysporum, F. solani, Moniliella suaveolens and T. harzianum (Table I) were common to both kinds of nests. The fungus garden is a dynamic system with a continuous supply of new substrate and disposal of the exhausted one. It was calculated that the material placed on the top of the fungus garden reaches its base anywhere from 5–7 weeks to a maximum of 4 months (Weber 1972; Fisher et al. 1996) and so it is reasonable to note that fungi are continuously introduced and excluded from this substrate. This may explain, e.g.,
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why so few species were found in common in both situations and why T. harzianum was the prevalent species in the field nests but isolated in less than 15 % of the laboratory nests. Table I. Fungi isolated from the fungus garden (G), the waste deposit (W) and fragments of the baits (B) from laboratory (n = 28) and field (n = 13) nests of Atta sexdens rubropilosa treated with Mirex-S® baitsa Laboratory nests
Species
Acremonium strictum Acremonium kiliense Arthrobotrys cladodes Aspergillus flavus Aspergillus niger var. niger Cladosporium cladosporioides Cunninghamella elegans Cunninghamella echinulata Escovopsis weberi Fusarium equiseti Fusarium oxysporum Fusarium solani Fusarium verticillioides Mariannaea elegans var. elegans Metarhizium anisopliae var. anisopliae Moniliella suaveolens Mucor hiemalis Mucor microsporus Penicillium citrinum Penicillium janthinellum Penicillium sp. subgen. Furcatum Piptocephalis sp. Syncephalastrum racemosum Trichoderma harzianum Trichoderma sp. Trichothecium roseum Mycelia sterila
Field nests
G
W
B
%
G
%
+ + + + – + + – + + – + – – – + – – + + – + + + – + +
– + – + – – + – – – + + + – – – – – + + + – – + – + –
– – – + – – – – – – – – – – – – – – + + – – – – – + –
4 14 4 18 0 4 18 0 21 4 4 18 4 0 0 4 0 0 18 11 4 7 54 14 0 14 4
+ + – – + – – + + – + + – + + + + + – – – – – + + – –
8 15 0 0 8 0 0 8 15 0 23 8 0 8 8 8 15 8 0 0 0 0 0 38 8 0 0
a% – percentage of sampled nests; (+) – the presence or (–) absence of fungi.
Despite the dynamics of the organic material and its influence in the diversity of the species, why were some strains regularly isolated from the fungus gardens? A possible explanation is that they are probably continuously introduced or perhaps some are strongly associated with this environment. As reported by Currie et al. (1999b), Escovopsis sp. is present in ≈43 % of the adult colonies of Atta sp. sampled in the Panama Canal region. According to the authors this proportion is lower in young nests. This fungus is considered to be a specialized parasite and can persist for a long time inside the fungus garden (Currie 2001). Our data confirm these results since this fungus was isolated in 21 and 15 % of the laboratory and field nests, respectively. The presence of the genus Trichoderma inside leaf-cutting ant nests was already recorded by Currie et al. (1999a) who considered this fungus a general pathogen for leaf-cutting ant colonies; however, the damage to the colonies is weak when compared with those done by Escovopsis sp. (Currie et al. 1999b). As far as we know there is no record of the occurrence of A. kiliense, F. oxysporum, and F. solani in leaf-cutting ant nests. It is known that these fungi, as T. harzianum, are saprotrophic species usually found in the soil (Domsch et al. 1980). At this moment we do not know if these species (as described by Currie et al. 1999b with regard to Trichoderma sp.), represent parasites of the nests or not. Still, F. oxysporum, T. harzianum, Trichothecium roseum, and Aspergillus flavus were isolated from the dead bodies of the workers discarded into the waste deposit of the laboratory nests. The occasional pathogen A. flavus was recorded on workers of Atta texana and Acromyrmex equinator (Lofgren et al. 1975; Hughes and Boomsma 2004) and this is the first report for the A. sexdens rubropilosa workers. The species S. racemosum, which was frequently isolated from the laboratory nests but not from the field nests, deserves special note. Commonly found in the soil (Domsch et al. 1980) it was expected to be found in the field colonies; however, this fungus was not present in the substrate used for rearing the labora-
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tory colonies since all the efforts to isolate it from these materials were unsuccessful. These observations lead to an interesting question: what are the factors that enable field colonies to successfully control this fungus but do not do it in the laboratory? Studies regarding the possible impact of S. racemosum on the field colonies should be carried out in order to determine if it could be a threat to the nests or not. We found two isolates of Cunninghamella elegans and one of C. echinulata. During the last 15 years, hundreds of leaf-cutting ants nests have been handled in our laboratory for different purposes and occasionally some nests have been contaminated with a cotton-like mycelium, which is initially white and gradually becomes light grey. This fungus was identified as C. elegans. If members of this genus are potential parasites of Atta nests or not, as pointed out before for A. kiliense, F. oxysporum, F. solani, and T. harzianum, is a matter for further study. In contrast with the findings of Currie et al. (1999b), our research showed that not only Escovopsis sp. can be the prevalent nonsymbiotic fungus in the leaf-cutting ant gardens but, in some circumstances, other species, such as S. racemosum and T. harzianum, can be frequently associated with the gardens of these insects. Currie et al. (2003b) suggest that Escovopsis sp. co-evolved together with the ants and their cultivars for millions of years and because of such natural history Escovopsis sp. strains have the capacity to overcome the defenses of the ants and to be “a step ahead” in the arms race of the ant–microbe symbiosis. Thus, it is possible that some of the species of fungi described here may represent strains in the process of adaptation to the environmental conditions of ant nests. We thank the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for supporting this research. 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