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Department of Ecology and Evolutionary Biology, 1200 Sunnyside Ave., University of Kansas, Lawrence, Kansas 66045
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ABSTRACT |
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Fungi in the genus Escovopsis are known only from the fungus gardens of attine ants. Previous work has established that these anamorphic fungi, allied with the Hypocreales, are specialized and potentially virulent parasites of the ancient mutualism between attine ants and their fungal cultivars. It is unclear whether the primary nutrient source for the pathogen is the mutualist fungal cultivar or the vegetative substrate placed on the gardens by the ants. Here, we determine whether Escovopsis weberi is a parasite of the fungal cultivar, a competitor for the leaf substrate, or both. Bioassays reveal that E. weberi exhibits rapid growth on pure cultivar and negligible growth on sterilized leaf fragments. Light microscopy examination of hyphalhyphal interactions between E. weberi and the ant fungal cultivar indicate that E. weberi, unlike invasive necrotrophs that always penetrate host hyphae, can secrete compounds that break down host mycelium before contact occurs. Thus, E. weberi is a necrotrophic parasite of the fungal cultivar of attine ants.
Key words: Acromyrmex, Atta, fungus-growing ants, mutualism, mycoparasite, necrotroph
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Chapela et al 1994). They provide fresh leaves and flowers as substrate to support the growth of the cultivar. The ants also promote the growth of their mutualistic fungus through pruning and the movement of enzymes within the garden (Martin 1970, 1987, Martin and Martin 1970, Bass and Cherrett 1996). They defend their fungal cultivar from invading microbes by grooming and licking the fungus garden and by removing (weeding) infected parts of the garden (Stahel and Giejskes 1939, Autuori 1941, Quinlan and Cherrett 1977, 1979, Currie and Stuart 2001).
Escovopsis is a genus of anamorphic fungi allied with the ascomycetous order Hypocreales (Currie et al 2003). It is commonly associated with the garden of fungus-growing ants, including the leaf-cutters, having been isolated in more than 50% of the gardens sampled in some studies (Currie et al 1999, Currie 2001b). Fungi in this genus appear to be obligately specialized on the attine ant-microbe symbiosis; they have been found growing naturally only in ant fungal gardens and associated dumps (Seifert et al 1995, Currie et al 1999, Bot et al 2001). Previous work has established that Escovopsis is a parasite of this mutualism (Currie et al 1999, Currie 2001b). The microfungus can limit the fitness of a colony and devastate a garden in a few days (Currie et al 1999; Currie 2001b, 2001a). Escovopsis also fulfills Koch’s postulates of pathogenicity; when it was isolated from diseased gardens and reapplied to healthy gardens, it caused the same disease (Currie et al 1999). Escovopsis, which does not produce airborne spores and apparently is not transmitted by founder queens, might be vectored by other invertebrates living in association with ant colonies (Currie et al 1999, Currie 2001a). In addition, it is an ancient member of the attine ant-microbe symbiosis, having co-evolved with the ants and their cultivar (Currie et al 2003).
While Escovopsis is clearly a parasite of the fungus garden, the precise nature of its pathogenicity is unknown. It might be a parasite of the cultivar, gaining nutrients from the mycelium of the basidiomycete; on the other hand, it might be a "weed", competing for the substrate placed on the gardens by the ants (Currie 2001a). In this study, we examined the question of whether E. weberi is a parasite of the cultivar of leaf-cutter ant gardens, a saprobe competing for the leaf substrate, or both. We used microscopy techniques and growth studies to determine E. weberi’s methods of mycoparasitism.
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MATERIALS AND METHODS |
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x 7 9/16 x 3
inches) placed on plastic polystyrene box "islands" (7 x 5
x 3 inches). These "islands" were kept on trays (21x 10
x 2inches) filled with soapy water to prevent movement of mites that could vector E. weberi spores among colonies.
To examine the ability of E. weberi (Muchovej and Della Lucia 1990) to grow on leaves and cultivar, we conducted bioassays on four different substrates, as well as on a nutrient-free control treatment. The five treatments were: (i) four pieces of the axenic cultivar (c), (ii) two pieces of the cultivar + two pieces of leaves manually cut (ccl), (iii) four pieces of manually cut leaves (cl), (iv) four pieces of leaves taken from ant colonies (al), and (v) no nutrient source added (control). All bioassays were conducted in 60 x 15 mm Petri plates on water agar (15 g agar/L water). The bottom of each plate was marked using a square stencil (2.1 x 2.1 cm). The substrate pieces were placed outside the corners of the square, and E. weberi was inoculated aseptically in the center of each plate (see FIG. 1). Five plates of each treatment were made to test E. weberi strains from each of the seven colonies, for a total of 175 plates (7 E. weberi isolates x 5 treatments x 5 replicates/treatment = 175). Isolates of the fungal cultivar and E. weberi were obtained by plating garden pieces on potato-dextrose agar (PDA) plates (see Currie et al 1999). E. weberi was tested against the cultivar from the garden from which it was isolated. Cultures of the fungi used in the experiments are stored at the National Museum of Natural History, Smithsonian Institution, under the aforementioned collection numbers. The leaves used for both the manual cutting treatment as well as the ant-cut leaves were from Redbud (Cercis canadensis). Manual cutting was done with a 3 mm cork borer, and the leaf fragments then were autoclaved. Leaf material taken from ant colonies was collected from the surface of fungus gardens, once the ants had masticated it. It was washed with alcohol and water to remove garden fungus and autoclaved. Although the leaf fragments were at the top of the garden and had not been fully integrated into the fungal matrix, visible cultivar hyphae were in the area, making it necessary to sterilize the leaf surfaces to ensure that, if E. weberi grew toward the leaves, it was not attracted to latent cultivar fragments. After surface sterilization, the leaves were autoclaved. To perform our experiments, we found it necessary to fully sterilize the leaves; surface sterilized leaves that were not autoclaved became overgrown with several types of fungi overnight when placed on water agar. Digital photographs were taken of each plate daily for 1 wk with a Nikon Coolpix 5000 camera. The photographs were examined to determine when E. weberi hyphae initially reached the pieces of substrate or the equivalent distance in the case of the control plates. Growth was quantified as the number of substrate pieces reached. Some plates became contaminated and thus were not used in the analyses. Because of contamination, the treatments each had different numbers of total substrate pieces. The numbers of plates that could be scored were: Acromyrmex octospinosus c = 15, ccl = 13, al = 13, cl = 14, control = 15; Atta colombica c = 16, ccl = 19, al = 17, cl = 12, control = 20, for a total of 154 plates and 616 substrate pieces. The data were analyzed using Chi-square tests (Steel and Torrie 1980).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and 2), causing them to turn yellow or brown and to shrivel. The parasite grew poorly in the absence of the cultivar, even when leaves were present. There was significantly higher growth in the presence of the cultivar than in the presence of leaves alone (
2 = 335.611, P < 0.001). When E. weberi was presented both with leaves and with cultivar, it typically grew toward the cultivar first and then spread over the rest of the plate (FIG. 1). There was no significant difference in the amount of growth of E. weberi from Atta colombica colonies and from Acromyrmex octospinosus colonies (2 = 1.124, P = 0.289). No growth of E. weberi was observed in the water-agar control treatments.
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). In seven of the slide cultures, the cultivar hyphae were not noticeably faded before contact with E. weberi. One of the slides became contaminated before contact between the two fungi could occur.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), further suggesting that it is not a competitor for the leaf material. Thus, our results indicate that E. weberi is a parasite of the fungal cultivar and not a weed.
There are two basic types of mycoparasites: biotrophs, which feed on the living host cytoplasm, and necrotrophs, which kill their hosts, then digest the dead biomass ( Jeffries and Young 1994). The discoloration and collapse of the cultivar mycelium in this experiment and the collapse of the ant gardens in experiments by Currie et al (1999) demonstrate that E. weberi kills the cultivar to gain nutrients from it and is therefore a necrotroph. Using light microscopy techniques, we observed that E. weberi in many cases was able to degrade cultivar hyphae from a distance of at least 25 µm; penetration was not necessary for parasitism to occur. We concluded that E. weberi is a contact necrotroph that does not have to penetrate the hyphae of its host, as opposed to an invasive necrotroph that must penetrate host tissue ( Jeffries and Young 1994).
Our finding that E. weberi is a necrotrophic parasite is not surprising perhaps because it is closely related to other necrotrophic parasites. The sister group of Escovopsis is the family Hypocreaceae (Currie et al 2003b), which includes many mycoparasitic fungi. For example, several species of Hypomyces are parasites of fungi in the family Boletaceae (Rogerson and Samuels 1989). The genus Trichoderma, an anamorph allied with the Hypocreaceae, includes ecologically dominant necrotrophic species, some of which parasitize the mushroom Agaricus bisporus ( Jeffries and Young 1994, Castle et al 1998, Samuels et al 2002). Trichoderma sp. are contact necrotrophs capable of using several types of antagonism: (i) long-range enzyme activity (Dennis and Webster 1971), (ii) hyphal interference involving close-range toxin (volatile and nonvolatile) production and coiling around host hyphae, and (iii) penetration (DeOliveira et al 1984, Jeffries and Young 1994). Perhaps Escovopsis spp., like Trichoderma spp., are capable of multiple types of antagonism; further work must be done to determine whether it is capable of coiling and/or penetration.
The fungal genus Escovopsis is a group of highly evolved obligate mycoparasites. It has an ancient origin within the attine ant-microbe mutualism and apparently has been co-evolving with the ants and their cultivar for more than 50 000 000 yr (Currie et al 2003). In this study, we established that E. weberi acts as a necrotrophic parasite on the cultivar of leaf-cutter ants, the most phylogenetically derived genera of fungus-growing ants. Future work should examine the activity of species of Escovopsis that infect the gardens of the more basal ant genera. Indeed, studies on the pathogenicity of Escovopsis spp. across the extant phylogenetic diversity of this ancient and host-specific association could lead to better understanding of the evolution of mycoparasitism in general. In addition, further studies on the mechanisms of Escovopsis pathogenicity might provide insights for using the fungus in biological control efforts for leaf-cutter ants, which are economically important pests in the Neotropics (Weber 1972).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 E-mail: htfreynolds{at}hotmail.com 
2 Corresponding author. E-mail: ccurrie{at}bact.wisc.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bass M, Cherrett JM. 1996. Leaf-cutting ants (Formicidae, Attini) prune their fungus to increase and direct its productivity. Funct Ecol 10:55–61.
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