Isolation and characterization of actinobacteria ectosymbionts from Acromyrmex subterraneus brunneus (Hymenoptera, Formicidae)

The ectosymbiont actinobacterium Pseudonocardia was isolated from the integument of Acromyrmex leaf-cutter ants and seems to play a crucial role in maintaining asepsis of the nest. Currently, there has been an intensive search for Pseudonocardia associated with several attine species, but few studies have indicated that other actinobacteria may be associated with these ants as well. We therefore characterized the culturable actinobacteria community associated with the integument of the fungus-growing ant Acromyrmex subterraneus brunneus Forel, 1893 (Hymenoptera: Formicidae). Ectosymbionts were isolated using four different media and characterized by morphological and molecular (16S rDNA) methods. A total of 20 strains were isolated, of which 17 were characterized as Streptomyces spp., and one isolate each as Pseudonocardia, Kitassatospora and Propionicimonas. Unlike other Acromyrmex species, A. subterraneus brunneus is associated with a diversity of actinobacteria. Even though Pseudonocardia is present on this leaf-cutting ant’s integument, the number and diversity of Streptomyces spp. found differs from those of previous studies with other attine ants and suggest that different culturing approaches are needed to characterize the true diversity of microbes colonizing the integument of attine ants. Moreover, understanding the diversity of the culturable actinobacteria associated with A. subterraneus brunneus should increase our knowledge of the evolutionary relationship of this intricate symbiotic association.     

Chemical warfare between fungus-growing ants and their pathogens

Fungus-growing attine ants are under constant threat from fungal pathogens such as the specialized mycoparasite Escovopsis, which uses combined physical and chemical attack strategies to prey on the fungal gardens of the ants. In defence, some species assemble protective microbiomes on their exoskeletons that contain antimicrobial-producing Actinobacteria. Underlying this network of mutualistic and antagonistic interactions are an array of chemical signals. Escovopsis weberi produces the shearinine terpene-indole alkaloids, which affect ant behaviour, diketopiperazines to combat defensive bacteria, and other small molecules that inhibit the fungal cultivar. Pseudonocardia and Streptomyces mutualist bacteria produce depsipeptide and polyene macrolide antifungals active against Escovopsis spp. The ant nest metabolome is further complicated by competition between defensive bacteria, which produce antibacterials active against even closely related species.     

Reduced biological control and enhanced chemical pest management in the evolution of fungus farming in ants

To combat disease, most fungus-growing ants (Attini) use antibiotics from mutualistic bacteria (Pseudonocardia) that are cultured on the ants'' exoskeletons and chemical cocktails from exocrine glands, especially the metapleural glands (MG). Previous work has hypothesized that (i) Pseudonocardia antibiotics are narrow-spectrum and control a fungus (Escovopsis) that parasitizes the ants'' fungal symbiont, and (ii) MG secretions have broad-spectrum activity and protect ants and brood. We assessed the relative importance of these lines of defence, and their activity spectra, by scoring abundance of visible Pseudonocardia for nine species from five genera and measuring rates of MG grooming after challenging ants with disease agents of differing virulence. Atta and Sericomyrmex have lost or greatly reduced the abundance of visible bacteria. When challenged with diverse disease agents, including Escovopsis, they significantly increased MG grooming rates and expanded the range of targets. By contrast, species of Acromyrmex and Trachymyrmex maintain abundant Pseudonocardia. When challenged, these species had lower MG grooming rates, targeted primarily to brood. More elaborate MG defences and reduced reliance on mutualistic Pseudonocardia are correlated with larger colony size among attine genera, raising questions about the efficacy of managing disease in large societies with chemical cocktails versus bacterial antimicrobial metabolites.     

Specificity and stability of the Acromyrmex–Pseudonocardia symbiosis

The stability of mutualistic interactions is likely to be affected by the genetic diversity of symbionts that compete for the same functional niche. Fungus‐growing (attine) ants have multiple complex symbioses and thus provide ample opportunities to address questions of symbiont specificity and diversity. Among the partners are Actinobacteria of the genus Pseudonocardia that are maintained on the ant cuticle to produce antibiotics, primarily against a fungal parasite of the mutualistic gardens. The symbiosis has been assumed to be a hallmark of evolutionary stability, but this notion has been challenged by culturing and sequencing data indicating an unpredictably high diversity. We used 454 pyrosequencing of 16S rRNA to estimate the diversity of the cuticular bacterial community of the leaf‐cutting ant Acromyrmex echinatior and other fungus‐growing ants from Gamboa, Panama. Both field and laboratory samples of the same colonies were collected, the latter after colonies had been kept under laboratory conditions for up to 10 years. We show that bacterial communities are highly colony‐specific and stable over time. The majority of colonies (25/26) had a single dominant Pseudonocardia strain, and only two strains were found in the Gamboa population across 17 years, confirming an earlier study. The microbial community on newly hatched ants consisted almost exclusively of a single strain of Pseudonocardia while other Actinobacteria were identified on older, foraging ants in varying but usually much lower abundances. These findings are consistent with recent theory predicting that mixtures of antibiotic‐producing bacteria can remain mutualistic when dominated by a single vertically transmitted and resource‐demanding strain.     

Coevolution between attine ants and actinomycete bacteria: a reevaluation

We reassess the coevolution between actinomycete bacteria and fungus-gardening (attine) ants. Actinomycete bacteria are of special interest because they are metabolic mutualists of diverse organisms (e.g., in nitrogen-fixation or antibiotic production) and because Pseudonocardia actinomycetes are thought to serve disease-suppressing functions in attine gardens. Phylogenetic information from culture-dependent and culture-independent microbial surveys reveals (1) close affinities between free-living and ant-associated Pseudonocardia, and (2) essentially no topological correspondence between ant and Pseudonocardia phylogenies, indicating frequent bacterial acquisition from environmental sources. Identity of ant-associated Pseudonocardia and isolates from soil and plants implicates these environments as sources from which attine ants acquire Pseudonocardia. Close relatives of Atta leafcutter ants have abundant Pseudonocardia, but Pseudonocardia in Atta is rare and appears at the level of environmental contamination. In contrast, actinomycete bacteria in the genera Mycobacterium and Microbacterium can be readily isolated from gardens and starter-cultures of Atta. The accumulated phylogenetic evidence is inconsistent with prevailing views of specific coevolution between Pseudonocardia, attine ants, and garden diseases. Because of frequent acquisition, current models of Pseudonocardia-disease coevolution now need to be revised. The effectiveness of Pseudonocardia antibiotics may not derive from advantages in the coevolutionary arms race with specialized garden diseases, as currently believed, but from frequent recruitment of effective microbes from environmental sources. Indeed, the exposed integumental structures that support actinomycete growth on attine ants argue for a morphological design facilitating bacterial recruitment. We review the accumulated evidence that attine ants have undergone modifications in association with actinomycete bacteria, but we find insufficient support for the reverse, modifications of the bacteria resulting from the interaction with attine ants. The defining feature of coevolution--reciprocal modification--therefore remains to be established for the attine ant-actinomycete mutualism.     

Disease defence through generations: leaf‐cutter ants and their symbiotic bacteria

Microbial ecology of animals is taking on significance in the modern dialogue for the biology of species. Similar to a nuclear genome, the entire bacterial assemblage maintains an ancestral signal of the host's evolution leading to cophylogeny between the host and the microbes they harbour (Brucker & Bordenstein 2012b). The stability of such associations is of great interest as they provide a means for species to acquire new traits and genetic diversity that their own genomes lack (McFall‐Ngai et al. 2013). The role of gut microbiota, for example, in host health and nutrition is widely recognized and a shared characteristic among animals. The role of bacteria colonizing the outside surfaces of animals is less well understood, but rather than random colonization, these microbes on skin, cuticles, scales and feathers in many cases provide benefits to the host. The symbiosis of leaf‐cutter ants, their fungus gardens and their microbiota is a fascinating and complex system. Whether culture‐independent bacterial diversity on the cuticle of leaf‐cutter ants is high or highly constrained by subcuticular gland secretions is one prominent question. In this issue of Molecular Ecology, Andersen et al. (2013) show that leaf‐cutting ants, Acromyrmex echinatior, maintain a dominant and colony‐specific bacterium called Pseudonocardia on their cuticles (the laterocervical plates in particular). This bacterium is involved in protecting the ants and their fungal gardens from disease. Other fungus‐gardening attine species as well as soil and vegetation can harbour Pseudonocardia. However, it was previously unknown how stable the bacterial strain–ant colony association was through the lifetime of the colony.     

Placement of attine ant-associated Pseudonocardia in a global Pseudonocardia phylogeny (Pseudonocardiaceae, Actinomycetales): a test of two symbiont-association models

We reconstruct the phylogenetic relationships within the bacterial genus Pseudonocardia to evaluate two models explaining how and why Pseudonocardia bacteria colonize the microbial communities on the integument of fungus-gardening ant species (Attini, Formicidae). The traditional Coevolution-Codivergence model views the integument-colonizing Pseudonocardia as mutualistic microbes that are largely vertically transmitted between ant generations and that supply antibiotics that specifically suppress the garden pathogen Escovopsis. The more recent Acquisition model views Pseudonocardia as part of a larger integumental microbe community that frequently colonizes the ant integument from environmental sources (e.g., soil, plant material). Under this latter model, ant-associated Pseudonocardia may have diverse ecological roles on the ant integument (possibly ranging from pathogenic, to commensal, to mutualistic) and are not necessarily related to Escovopsis suppression. We test distinct predictions of these two models regarding the phylogenetic proximity of ant-associated and environmental Pseudonocardia. We amassed 16S-rRNA gene sequence information for 87 attine-associated and 238 environmental Pseudonocardia, aligned the sequences with the help of RNA secondary structure modeling, and reconstructed phylogenetic relationships using a maximum-likelihood approach. We present 16S-rRNA secondary structure models of representative Pseudonocardia species to improve sequence alignments and identify sequencing errors. Our phylogenetic analyses reveal close affinities and even identical sequence matches between environmental Pseudonocardia and ant-associated Pseudonocardia, as well as nesting of environmental Pseudonocardia in subgroups that were previously thought to be specialized to associate only with attine ants. The great majority of ant-associated Pseudonocardia are closely related to autotrophic Pseudonocardia and are placed in a large subgroup of Pseudonocardia that is known essentially only from cultured isolates (rather than cloned 16S sequences). The preponderance of the known ant-associated Pseudonocardia in this latter clade of culturable lineages may not necessarily reflect abundance of these Pseudonocardia types on the ants, but isolation biases when screening for Pseudonocardia (e.g., preferential isolation of autotrophic Pseudonocardia with minimum-nutrient media). The accumulated phylogenetic patterns and the possibility of isolation biases in previous work further erode support for the traditional Coevolution-Codivergence model and calls for continued revision of our understanding how and why Pseudonocardia colonize the microbial communities on the integument of fungus-gardening ant species.     

A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in ～30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.     

Phylogenetic patterns of ant–fungus associations indicate that farming strategies,not only a superior fungal cultivar,explain the ecological success of leafcutter ants

To elucidate fungicultural specializations contributing to ecological dominance of leafcutter ants, we estimate the phylogeny of fungi cultivated by fungus‐growing (attine) ants, including fungal cultivars from (i) the entire leafcutter range from southern South America to southern North America, (ii) all higher‐attine ant lineages (leafcutting genera Atta, Acromyrmex; nonleafcutting genera Trachymyrmex, Sericomyrmex) and (iii) all lower‐attine lineages. Higher‐attine fungi form two clades, Clade‐A fungi (Leucocoprinus gongylophorus, formerly Attamyces) previously thought to be cultivated only by leafcutter ants, and a sister clade, Clade‐B fungi, previously thought to be cultivated only by Trachymyrmex and Sericomyrmex ants. Contradicting this traditional view, we find that (i) leafcutter ants are not specialized to cultivate only Clade‐A fungi because some leafcutter species ranging across South America cultivate Clade‐B fungi; (ii) Trachymyrmex ants are not specialized to cultivate only Clade‐B fungi because some Trachymyrmex species cultivate Clade‐A fungi and other Trachymyrmex species cultivate fungi known so far only from lower‐attine ants; (iii) in some locations, single higher‐attine ant species or closely related cryptic species cultivate both Clade‐A and Clade‐B fungi; and (iv) ant–fungus co‐evolution among higher‐attine mutualisms is therefore less specialized than previously thought. Sympatric leafcutter ants can be ecologically dominant when cultivating either Clade‐A or Clade‐B fungi, sustaining with either cultivar‐type huge nests that command large foraging territories; conversely, sympatric Trachymyrmex ants cultivating either Clade‐A or Clade‐B fungi can be locally abundant without achieving the ecological dominance of leafcutter ants. Ecological dominance of leafcutter ants therefore does not depend primarily on specialized fungiculture of L. gongylophorus (Clade‐A), but must derive from ant–fungus synergisms and unique ant adaptations.     

The molecular phylogenetics of Trachymyrmex Forel ants and their fungal cultivars provide insights into the origin and coevolutionary history of ‘higher‐attine’ ant agriculture

The fungus‐growing ants and their fungal cultivars constitute a classic example of a mutualism that has led to complex coevolutionary dynamics spanning c. 55–65 Ma. Of the five agricultural systems practised by fungus‐growing ants, higher‐attine agriculture, of which leaf‐cutter agriculture is a derived subset, remains poorly understood despite its relevance to ecosystem function and human agriculture across the Neotropics and parts of North America. Among the ants practising higher‐attine agriculture, the genus Trachymyrmex Forel, as currently defined, shares most‐recent common ancestors with both the leaf‐cutter ants and the higher‐attine genera Sericomyrmex Mayr and Xerolitor Sosa‐Calvo et al. Although previous molecular‐phylogenetic studies have suggested that Trachymyrmex is a paraphyletic grade, until now insufficient taxon sampling has prevented a full investigation of the evolutionary history of this group and limited the possibility of resolving its taxonomy. Here we describe the results of phylogenetic analyses of 38 Trachymyrmex species, including 27 of the 49 described species and at least 11 new species, using four nuclear markers, as well as phylogenetic analyses of the fungi cultivated by 23 species of Trachymyrmex using two markers. We generated new genetic data for 112 ants (402 new gene sequences) and 95 fungi (153 new gene sequences). Our results corroborate previous findings that Trachymyrmex, as currently defined, is paraphyletic. We propose recognizing two new genera, Mycetomoellerius gen.n. and Paratrachymyrmex gen.n. , and restricting the continued use of Trachymyrmex to the clade of nine largely North American species that contains the type species [Trachymyrmex septentrionalis (McCook)] and that is the sister group of the leaf‐cutting ants. Our fungal cultivar phylogeny generally corroborates previously observed broad patterns of ant–fungus association, but it also reveals further violations of those patterns. Higher‐attine fungi are divided into two groups: (i) the single species Leucoagaricus gongylophorus (Möller); and (ii) its sister clade, consisting of multiple species, recently referred to as Leucoagaricus Singer ‘clade B’. Our phylogeny indicates that, although most non‐leaf‐cutting higher‐attine ants typically cultivate species in clade B, some species cultivate L. gongylophorus, whereas still others cultivate fungi typically associated with lower‐attine agriculture. This indicates that the attine agricultural systems, which are currently defined by associations between ants and fungi, are not entirely congruent with ant and fungal phylogenies. They may, however, be correlated with as yet poorly understood biological traits of the ants and/or of their microbiomes.     

Two heterologously expressed <Emphasis Type="Italic">Planobispora rosea</Emphasis> proteins cooperatively induce <Emphasis Type="Italic">Streptomyces lividans</Emphasis> thiostrepton uptake and storage from the extracellular medium

Background  

A bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli - Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics.     

Co‐evolutionary patterns and diversification of ant–fungus associations in the asexual fungus‐farming ant Mycocepurus smithii in Panama

Partner fidelity through vertical symbiont transmission is thought to be the primary mechanism stabilizing cooperation in the mutualism between fungus‐farming (attine) ants and their cultivated fungal symbionts. An alternate or additional mechanism could be adaptive partner or symbiont choice mediating horizontal cultivar transmission or de novo domestication of free‐living fungi. Using microsatellite genotyping for the attine ant Mycocepurus smithii and ITS rDNA sequencing for fungal cultivars, we provide the first detailed population genetic analysis of local ant–fungus associations to test for the relative importance of vertical vs. horizontal transmission in a single attine species. M. smithii is the only known asexual attine ant, and it is furthermore exceptional because it cultivates a far greater cultivar diversity than any other attine ant. Cultivar switching could permit the ants to re‐acquire cultivars after garden loss, to purge inferior cultivars that are locally mal‐adapted or that accumulated deleterious mutations under long‐term asexuality. Compared to other attine ants, symbiont choice and local adaptation of ant–fungus combinations may play a more important role than partner‐fidelity feedback in the co‐evolutionary process of M. smithii and its fungal symbionts.     

Production of antibiotics and enzymes by soil microorganisms from the windmill islands region, Wilkes Land, East Antarctica

The distribution of microorganisms in four soil samples taken near Casey Station, Wilkes Land, and on Dewart Island, Frazier Islands (East Antarctica), was studied using isolation cultures at different temperatures. The fungal assemblages comprised Penicillium, Alternaria, Cladosporium, Phoma, Verticillium, Phialophora, Candida and Rhodotorula. Microalgal Chlorophyta were also common. Among the bacteria representatives of cyanobacteria, Staphylococcus, Bacillus, Pantoea, Streptomyces, Micromonospora, and coryne- and nocardioform species were found. Some of the isolated actinomycete strains, determined as Arthrobacter, Rhodococcus, Streptomyces and Micromonospora, produced extracellular substances with antibacterial and antifungal activities. Their potential as biological agents against phytopathogenic bacteria and fungi is discussed.     

<Emphasis Type="Italic">Pseudonocardia bannaensis</Emphasis> sp. nov., a novel actinomycete isolated from the surface-sterilized roots of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

During the course of our research on new actinobacterial sources, a novel actinomycete strain YIM 63101^T was isolated from the surface-sterilized roots of Artemisia annua L. collected from Xishuangbanna, Yunnan province, south-west China and characterized by using a polyphasic approach. The strain formed well-differentiated aerial and substrate mycelia. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 63101^T belongs to the genus Pseudonocardia, with highest similarity to “Pseudonocardia artemisiae YIM 63587^T” (99.4%). Sequence similarities between strain YIM 63101^T and the other Pseudonocardia species ranged from 97.0 (Pseudonocardia saturnea IMSNU 20052^T) to 94.0% (Pseudonocardia compacta IMSNU 20111^T). The chemotaxonomic characteristics, such as cell wall diaminopimelic acid, whole-cell sugars, fatty acid components and the major menaquinones suggested that the organism belonged to the genus Pseudonocardia. The G + C content of the genomic DNA was 69.4 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA–DNA hybridization results, it is proposed that strain YIM 63101^T represents a novel species of the genus Pseudonocardia, named Pseudonocardia bannaensis sp. nov. The type strain is YIM 63101^T (= CCTCC AA 208077 ^T = DSM 45300^T).     

Planting of fungus onto hibernating workers of the fungus-growing ant Mycetosoritis clorindae (Attini, Formicidae)

We describe a peculiar fungus-coating behavior of the attine ant Mycetosoritis clorindae, where workers plant fungal mycelium on hibernating nestmates. Hibernating nestmates become ultimately enveloped in a live mycelial coat, remain motionless in this coated state, and essentially become integrated into the garden matrix. The shallow nest architecture of M. clorindae (depth of main garden is 15–30 cm) in southern Brazil forces the ants to overwinter at relatively low temperatures in the topmost soil layer. Fungal coating may help the ants to survive the prolonged periods of immobility during winter. Fungus-planting on attine adults is so far unknown from other attine species, but the behavior parallels the planting of mycelium on larvae and pupae occurring in many attine species. Planting of mycelium on adult nestmates may have been overlooked so far in attine ants because this behavior may occur only in dormant nests, which are least frequently collected. The possible adaptive functions of fungus coatings of hibernating adults and developing brood are likely similar, including for example physical protection, prevention of desiccation, shielding against parasites and predators (e.g., army ants), or defense against diseases.     

Phenotypic and phylogenetic characterization of actinomycetes isolated from <Emphasis Type="Italic">Lasius niger</Emphasis> and <Emphasis Type="Italic">Formica cunicularia</Emphasis> ants

Multiple actinomycete strains were isolated from two ant species, Lasius niger and Formica cunicularia, and their phenotypic properties and phylogenetic position were studied. Partial sequencing of 16S rRNA assigned the greater part of them to the genus Streptomyces, but only one belonged to Nocardia. However, some isolates had significant color and morphological differences from their closest phylogenetic relatives. The abundance and biodiversity of actinomycete communities isolated from L. niger ants greatly exceeded those found for F. cunicularia. All of the actinomycetes associated with F. cunicularia ants demonstrated cellulolytic activity, but only one had such ability among the strains associated with black ants.     

<Emphasis Type="Italic">Pseudonocardia serianimatus</Emphasis> sp. nov., a novel actinomycete isolated from the surface-sterilized leaves of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

An aerobic, non-motile, catalase-positive, Gram-stain positive actinomycete designated YIM 63233^T was isolated from the surface-sterilized leaves of Artemisia annua L. and characterized using a polyphasic taxonomic approach. Optimal growth occurred at 20–28°C, pH 6.0–7.0 and in the presence of 0–3% (w/v) NaCl. 16S rRNA gene sequence-based phylogenetic analysis showed that strain YIM 63233^T clustered with species of the genus Pseudonocardia, displaying ≥1.2% sequence divergence with recognized species of this genus (from 98.8 to 94.0%). Relatively low levels of DNA–DNA relatedness were found between strain YIM 63233^T and Pseudonocardia petroleophila IMSNU 22072^T, which supported the classification of strain YIM 63233^T within a novel species of the genus Pseudonocardia. The G + C content of genomic DNA was 72.0 mol%. Strain YIM 63233^T possessed chemotaxonomic markers that were consistent with classification in the genus Pseudonocardia, i.e. the predominant fatty acids were iso-C16:0 (32.27%), C16:0 10-methyl (8.73%) and C17:1ω8c (8.30%), whilst the predominant menaquinone was MK-8(H₄). The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. The major cell wall sugars were glucose, arabinose, galactose, mannose and rhamnose. The results of physiological and biochemical tests and DNA–DNA hybridization allowed the phenotypic and genotypic differentiation of strain YIM 63233^T from its closest phylogenetic neighbours. Therefore, the new isolate YIM 63233^T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia serianimatus sp. nov. is proposed. The type strain is YIM 63233^T (=DSM 45302^T = CCTCC AA 208079^T).     

Fungus-growing Allomerus ants are associated with antibiotic-producing actinobacteria

Fungus-growing attine ants use natural-product antibiotics produced by mutualist actinobacteria as ‘weedkillers’ in their fungal gardens. Here we report for the first time that fungus-growing Allomerus ants, which lie outside the tribe Attini, are associated with antifungal-producing actinobacteria, which offer them protection against non-cultivar fungi isolated from their ant-plants.     

Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants

We investigate the diversity of yeasts isolated in gardens of the leafcutter ant Atta texana. Repeated sampling of gardens from four nests over a 1-year time period showed that gardens contain a diverse assemblage of yeasts. The yeast community in gardens consisted mostly of yeasts associated with plants or soil, but community composition changed between sampling periods. In order to understand the potential disease-suppressing roles of the garden yeasts, we screened isolates for antagonistic effects against known microfungal garden contaminants. In vitro assays revealed that yeasts inhibited the mycelial growth of two strains of Escovopsis (a specialized attine garden parasite), Syncephalastrum racemosum (a fungus often growing in gardens of leafcutter lab nests), and the insect pathogen Beauveria bassiana. These garden yeasts add to the growing list of disease-suppressing microbes in attine nests that may contribute synergistically, together with actinomycetes and Burkholderia bacteria, to protect the gardens and the ants against diseases. Additionally, we suggest that garden immunity against problem fungi may therefore derive not only from the presence of disease-suppressing Pseudonocardia actinomycetes, but from an enrichment of multiple disease-suppressing microorganisms in the garden matrix.