Biogeography of mutualistic fungi cultivated by leafcutter ants.

Leafcutter ants propagate co-evolving fungi for food. The nearly 50 species of leafcutter ants (Atta, Acromyrmex) range from Argentina to the United States, with the greatest species diversity in southern South America. We elucidate the biogeography of fungi cultivated by leafcutter ants using DNA sequence and microsatellite-marker analyses of 474 cultivars collected across the leafcutter range. Fungal cultivars belong to two clades (Clade-A and Clade-B). The dominant and widespread Clade-A cultivars form three genotype clusters, with their relative prevalence corresponding to southern South America, northern South America, Central and North America. Admixture between Clade-A populations supports genetic exchange within a single species, Leucocoprinus gongylophorus. Some leafcutter species that cut grass as fungicultural substrate are specialized to cultivate Clade-B fungi, whereas leafcutters preferring dicot plants appear specialized on Clade-A fungi. Cultivar sharing between sympatric leafcutter species occurs frequently such that cultivars of Atta are not distinct from those of Acromyrmex. Leafcutters specialized on Clade-B fungi occur only in South America. Diversity of Clade-A fungi is greatest in South America, but minimal in Central and North America. Maximum cultivar diversity in South America is predicted by the Kusnezov-Fowler hypothesis that leafcutter ants originated in subtropical South America and only dicot-specialized leafcutter ants migrated out of South America, but the cultivar diversity becomes also compatible with a recently proposed hypothesis of a Central American origin by postulating that leafcutter ants acquired novel cultivars many times from other nonleafcutter fungus-growing ants during their migrations from Central America across South America. We evaluate these biogeographic hypotheses in the light of estimated dates for the origins of leafcutter ants and their cultivars.

Rhodosporidiobolus geoffroeae sp. nov., a basidiomycetous yeast isolated from the waste deposit of the attine ant Acromyrmex lundii.

A novel basidiomycetous yeast was isolated from the waste deposit of the attine ant Acromyrmex lundii (Hymenoptera: Formicidae). The field colony was located in Santurce town, Santa Fe province, Argentina. The description of the novel species was based on strain LLU043T. Analysis of the D1/D2 domains of the LSU rRNA gene sequences in GenBank demonstrated that strain LLU043T, belongs to the Rhodosporidiobolus clade and is closely related to Rhodosporidiobolus lusitaniae and Rhodosporidioboluscolostri with 97 % similarity to the two species. The novel species differs from R. lusitaniae and R. colostri in some physiological characteristics such as the lack of assimilation of cellobiose, salicin, succinate, citrate and ethylamine. The name Rhodosporidiobolus geoffroeae sp. nov. is proposed, with LLU043T (=CBS 12828T=CBMAI 1618T) as the type strain.

Wickerhamomyces spegazzinii sp. nov., an ascomycetous yeast isolated from the fungus garden of Acromyrmex lundii nest (Hymenoptera: Formicidae).

A novel ascomycetous yeast species in the genus Wickerhamomyces was isolated from the fungus garden of an attine ant nest, Acromyrmex lundii (Hymenoptera: Formicidae), from Santa Fe province, Argentina. Pairwise sequence alignment of D1/D2 sequences in the GenBank (http://www.ncbi.nlm.nih.gov) database revealed that the novel species is related most closely to Wickerhamomyces subpelliculosus, Wickerhamomyces linferdii, Wickerhamomyces anomalus, Wickerhamomyces siamensis and Wickerhamomycesciferrii with 96% similarity to the first four. The species name Wickerhamomyces spegazzinii sp. nov. is proposed to accommodate this novel strain, which differs from the above species in melibiose, 5-keto-D-gluconate, succinate, and DL-lactate assimilation among others. The type strain is JLU025T (=CBS 12756T=CBMAI 1619T).

Screening of Yeasts for Selection of Potential Strains and Their Utilization for In Situ Microbial Detoxification (ISMD) of Sugarcane Bagasse Hemicellulosic Hydrolysate.

Many toxic compounds are produced and released in the hemicellulosic hydrolyzates during the acid pretreatment step, which are required for the disruption of the lignocelluloses matrix and sugars release. The conventional methods of detoxification i.e. overliming, activated charcoal, ion exchange or even membrane-based separations have the limitations in removal of these toxic inhibitors in fermentation process. Hence, it is imperative to explore biological methods to overcome the inhibitors by minimizing the filtration steps, sugar loss and chemical additions. In the present study we screened sixty-four strains of yeasts to select potential strains for detoxification of furfural, acetic acid, ferulic acid, 5-hydroxymethyl furfural (5-HMF) as carbon and energy source. Among these strains Pichia occidentalis M1, Y1'a, Y1'b and Y3' showed a significant decrease in the toxic compounds but we selected two best yeast strains i.e. P. occidentalis Y1'a and P. occidentalis M1 for the further experiments with an aim to remove the fermentation inhibitors. The yeasts P. occidentalis Y1'a and P. occidentalis M1 were grown aerobically in sugarcane bagasse hemicellulose hydrolysate under submerged cultivation. For each yeast, a 2(2) full factorial design was performed considering the variables-pH (4.0 or 5.0) and agitation rate (100 or 300 rpm), and the percentage removal of HMF, furfural, acetic acid and phenols from hemicellulosic hydrolysates were responsive variables. After 96 h of biological treatment, P. occidentalis M1 and P. occidentalis Y1'a showed 42.89 and 46.04 % cumulative removal of inhibitors, respectively.

Yamadazyma riverae sp. nov., a yeast species isolated from plant materials.

Nine strains of a novel yeast species were isolated from rotting wood, tree bark, ant nests or living as endophytes in leaves of Vellozia gigantea. Analysis of the sequences of the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit rRNA gene showed that this species was related to Candida insectorum in the Yamadazyma clade. The novel species differed from closely related species by 10 and 11 substitutions in the ITS region and the D1/D2 domains of the large subunit of the rRNA gene, respectively. The species is heterothallic and forms asci with one to two hat-shaped ascospores. The name Yamadazyma riverae sp. nov. is proposed for the novel species. The type strain is UFMG-CM-Y444T ( = CBS 14121T) and the allotype strain is TT12 ( = CBS 14098 = UFMG-CM-Y577). The Mycobank number is MB 813221.

Escovopsis trichodermoides sp. nov., isolated from a nest of the lower attine ant Mycocepurus goeldii.

Currently, five species are formally described in Escovopsis, a specialized mycoparasitic genus of fungus gardens of attine ants (Hymenoptera: Formicidae: tribe Attini). Four species were isolated from leaf-cutting ants in Brazil, including Escovopsis moelleri and Escovopsis microspora from nests of Acromyrmex subterraneus molestans, Escovopsis weberi from a nest of Atta sp. and Escovopsis lentecrescens from a nest of Acromyrmex subterraneus subterraneus. The fifth species, Escovopsis aspergilloides was isolated from a nest of the higher attine ant Trachymyrmex ruthae from Trinidad. Here, we describe a new species, Escovopsis trichodermoides isolated from a fungus garden of the lower attine ant Mycocepurus goeldii, which differs from the five other species by highly branched, trichoderma-like conidiophores lacking swollen vesicles, with reduced conidiogenous cells and distinctive conidia morphology. Phylogenetic analyses based on partial tef1 gene sequences support the distinctiveness of this species. A portion of the internal transcribed spacers of the nuclear rDNA was sequenced to serve as a DNA barcode. Future molecular and morphological studies in this group of fungi will certainly unravel the taxonomic diversity of Escovopsis associated with fungus-growing ants.

Hanseniaspora nectarophila sp. nov., a yeast species isolated from ephemeral flowers.

Seven apiculate yeast strains that were isolated from the flowers of Syphocampylus corymbiferus Pohl in Brazil are genetically, morphologically and phenotypically distinct from recognized species of the genera Hanseniaspora and Kloeckera. Genetic discontinuities between the novel strains and their closest relatives were found using a networking approach based on the concatenated sequences of the rRNA gene (internal transcribed spacer and D1/D2 of the LSU), and the protein-coding genes for actin and translation elongation factor-1α. Phylogenetic analysis based on the rRNA and the actin gene placed the novel species represented by the strains in close relationship to Hanseniaspora meyeri and Hanseniaspora clermontiae. PCR fingerprinting with microsatellite primers confirmed the genetic heterogeneity of the novel species. The name Hanseniaspora nectarophila sp. nov. is proposed, with UFMG POG a.1(T) ( = ZIM 2311(T)  = CBS 13383(T)) as the type strain; MycoBank no. MB807210. As the current description of the genus does not allow the presence of multilateral budding, an emended diagnosis of the genus Hanseniaspora Zikes is proposed.

Starmerella aceti f.a., sp. nov., an ascomycetous yeast species isolated from fungus garden of the leafcutter ant Acromyrmex balzani.

A novel yeast species was recovered from the fungus garden of the leaf-cutting ant Acromyrmex balzani (Hymenoptera: Formicidae). The growth of the novel yeast species is limited by its ability to metabolize only a few carbon and nitrogenous compounds. A remarkable characteristic of this strain is the vigorous growth in 1 % acetic acid. Sequence analysis of the D1/D2 domains of the LSU rRNA gene showed that the novel species belongs to the Starmerella clade and is phenotypically and genetically divergent from currently recognized species in this clade. Described here as Starmerella aceti f.a., sp. nov., it differs by 37 nucleotide substitutions in the D1/D2 region from Starmerella jinningensis CBS 11864(T), the most closely related species. The type strain of Starmerella aceti sp. nov. is TO 125(T) ( = CBMAI 1594(T) = CBS 13086(T)).

Fungal communities in the garden chamber soils of leaf-cutting ants.

Leaf-cutting ants modify the properties of the soil adjacent to their nests. Here, we examined whether such an ant-altered environment impacts the belowground fungal communities. Fungal diversity and community structure of soil from the fungus garden chambers of Atta sexdens rubropilosa and Atta bisphaerica, two widespread leaf-cutting ants in Brazil, were determined and compared with non-nest soils. Culture-dependent methods revealed similar species richness but different community compositions between both types of soils. Penicillium janthinellum and Trichoderma spirale were the prevalent isolates in fungus chamber soils and non-nest soils, respectively. In contrast to cultivation methods, analyses of clone libraries based on the internal transcribed spacer (ITS) region indicated that richness of operational taxonomic units significantly differed between soils of the fungus chamber and non-nest soils. FastUnifrac analyses based on ITS sequences further revealed a clear distinction in the community structure between both types of soils. Plectania milleri and an uncultured Clavariaceae fungus were prevalent in fungus chamber soils and non-nest soils, respectively. FastUnifrac analyses also revealed that fungal community structures of soil from the garden chambers markedly differed among ant species. Our findings suggest that leaf-cutting ants affect fungal communities in the soil from the fungus chamber in comparison to non-nest soils.

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.

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants.

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants' symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes' exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant-microbial symbiosis is discussed.

Leaf-cutting ant faecal fluid and mandibular gland secretion: effects on microfungi spore germination.

The mandibular gland secretion (MGS) and the faecal fluid (FF) of the leaf-cutting ant Atta sexdens rubropilosa Forel affected the spore germination of selected microfungi isolated from nests of this insect. MGS was more effective than the FF, completely inhibiting the spore germination of four out of six microfungi species.

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista.

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: Polybia-MPI: I D W K K L L D A A K Q I L-NH2 (1654.09 Da), Polybia-CP: I L G T I L G L L K S L-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.

Activity of Ricinus communis (Euphorbiaceae) and ricinine against the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae) and the symbiotic fungus Leucoagaricus gongylophorus.

The focus of this study was the identification of compounds from plant extracts for use in crop protection. This paper reports on the toxic activity of fractions of leaf extracts of Ricinus communis L (Euphorbiaceae) and isolated active compounds in the leaf-cutting ant Atta sexdens rubropilosa Forel and its symbiotic fungus Leucoagaricus gongylophorus (Singer) Möller. The main compounds responsible for activity against the fungus and ant in leaf extracts of R communis were found to be fatty acids for the former and ricinine for the ants.

A Brazilian population of the asexual fungus-growing ant Mycocepurus smithii (Formicidae, Myrmicinae, Attini) cultivates fungal symbionts with gongylidia-like structures.

Attine ants cultivate fungi as their most important food source and in turn the fungus is nourished, protected against harmful microorganisms, and dispersed by the ants. This symbiosis evolved approximately 50-60 million years ago in the late Paleocene or early Eocene, and since its origin attine ants have acquired a variety of fungal mutualists in the Leucocoprineae and the distantly related Pterulaceae. The most specialized symbiotic interaction is referred to as "higher agriculture" and includes leafcutter ant agriculture in which the ants cultivate the single species Leucoagaricus gongylophorus. Higher agriculture fungal cultivars are characterized by specialized hyphal tip swellings, so-called gongylidia, which are considered a unique, derived morphological adaptation of higher attine fungi thought to be absent in lower attine fungi. Rare reports of gongylidia-like structures in fungus gardens of lower attines exist, but it was never tested whether these represent rare switches of lower attines to L. gonglyphorus cultivars or whether lower attine cultivars occasionally produce gongylidia. Here we describe the occurrence of gongylidia-like structures in fungus gardens of the asexual lower attine ant Mycocepurus smithii. To test whether M. smithii cultivates leafcutter ant fungi or whether lower attine cultivars produce gongylidia, we identified the M. smithii fungus utilizing molecular and morphological methods. Results shows that the gongylidia-like structures of M. smithii gardens are morphologically similar to gongylidia of higher attine fungus gardens and can only be distinguished by their slightly smaller size. A molecular phylogenetic analysis of the fungal ITS sequence indicates that the gongylidia-bearing M. smithii cultivar belongs to the so-called "Clade 1"of lower Attini cultivars. Given that M. smithii is capable of cultivating a morphologically and genetically diverse array of fungal symbionts, we discuss whether asexuality of the ant host maybe correlated with low partner fidelity and active symbiont choice between fungus and ant mutualists.

Broad Escovopsis-inhibition activity of Pseudonocardia associated with Trachymyrmex ants.

Attine ants maintain an association with antibiotic-producing Actinobacteria found on their integuments. Evidence supports these bacteria as auxiliary symbionts that help ants to defend the fungus gardens against pathogens. Using Pseudonocardia strains isolated from Trachymyrmex ants, we tested whether the inhibitory capabilities of such strains are restricted to Escovopsis parasites that infect gardens of this ant genus. Twelve Pseudonocardia strains were tested in in vitro bioassays against Escovopsis strains derived from fungus gardens of Trachymyrmex (n = 1) and leaf-cutting ants (n = 3). Overall, significant differences were observed in the mycelial growth among each Escovopsis strain in the presence of Pseudonocardia. Particularly, Escovopsis from Acromyrmex and Trachymyrmex were the most inhibited strains in comparison to Escovopsis isolated from Atta. This result suggests that Pseudonocardia isolated from Trachymyrmex possibly secrete antimicrobial compounds effective against diverse Escovopsis strains. The fact that Trachymyrmex ants harbour Pseudonocardia strains with broad spectrum of activity and its defensive role on attine gardens are discussed.

Molecular and kinetic characterization of two extracellular Xylanases isolated from Leucoagaricus gongylophorus.

In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 °C, optimum pH between 8.5 and 10.5, and Km = 14.7 ± 7.6 mg mL(-1). Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 °C, and Km = 2.2 ± 0.5 mg mL(-1). XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 °C) and pH (4.0), as well as the kinetic parameters (Km = 7.4 ± 2.0 mg mL(-1)) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.

Multi-scale structural and chemical analysis of sugarcane bagasse in the process of sequential acid-base pretreatment and ethanol production by Scheffersomyces shehatae and Saccharomyces cerevisiae.

BACKGROUND: Heavy usage of gasoline, burgeoning fuel prices, and environmental issues have paved the way for the exploration of cellulosic ethanol. Cellulosic ethanol production technologies are emerging and require continued technological advancements. One of the most challenging issues is the pretreatment of lignocellulosic biomass for the desired sugars yields after enzymatic hydrolysis. We hypothesized that consecutive dilute sulfuric acid-dilute sodium hydroxide pretreatment would overcome the native recalcitrance of sugarcane bagasse (SB) by enhancing cellulase accessibility of the embedded cellulosic microfibrils. RESULTS: SB hemicellulosic hydrolysate after concentration by vacuum evaporation and detoxification showed 30.89 g/l xylose along with other products (0.32 g/l glucose, 2.31 g/l arabinose, and 1.26 g/l acetic acid). The recovered cellulignin was subsequently delignified by sodium hydroxide mediated pretreatment. The acid-base pretreated material released 48.50 g/l total reducing sugars (0.91 g sugars/g cellulose amount in SB) after enzymatic hydrolysis. Ultra-structural mapping of acid-base pretreated and enzyme hydrolyzed SB by microscopic analysis (scanning electron microcopy (SEM), transmitted light microscopy (TLM), and spectroscopic analysis (X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Fourier transform near-infrared (FT-NIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy) elucidated the molecular changes in hemicellulose, cellulose, and lignin components of bagasse. The detoxified hemicellulosic hydrolysate was fermented by Scheffersomyces shehatae (syn. Candida shehatae UFMG HM 52.2) and resulted in 9.11 g/l ethanol production (yield 0.38 g/g) after 48 hours of fermentation. Enzymatic hydrolysate when fermented by Saccharomyces cerevisiae 174 revealed 8.13 g/l ethanol (yield 0.22 g/g) after 72 hours of fermentation. CONCLUSIONS: Multi-scale structural studies of SB after sequential acid-base pretreatment and enzymatic hydrolysis showed marked changes in hemicellulose and lignin removal at molecular level. The cellulosic material showed high saccharification efficiency after enzymatic hydrolysis. Hemicellulosic and cellulosic hydrolysates revealed moderate ethanol production by S. shehatae and S. cerevisiae under batch fermentation conditions.

Foraging of Psilocybe basidiocarps by the leaf-cutting ant Acromyrmex lobicornis in Santa Fé, Argentina.

BACKGROUND: It is generally accepted that material collected by leaf-cutting ants of the genus Acromyrmex consists solely of plant matter, which is used in the nest as substrate for a symbiotic fungus providing nutrition to the ants. There is only one previous report of any leaf-cutting ant foraging directly on fungal basidiocarps. FINDINGS: Basidiocarps of Psilocybe coprophila growing on cow dung were actively collected by workers of Acromyrmex lobicornis in Santa Fé province, Argentina. During this behaviour the ants displayed typical signals of recognition and continuously recruited other foragers to the task. Basidiocarps of different stages of maturity were being transported into the nest by particular groups of workers, while other workers collected plant material. CONCLUSIONS: The collection of mature basidiocarps with viable spores by leaf-cutting ants in nature adds substance to theories relating to the origin of fungiculture in these highly specialized social insects.

Anti-Candida properties of urauchimycins from actinobacteria associated with trachymyrmex ants.

After decades of intensive searching for antimicrobial compounds derived from actinobacteria, the frequency of isolation of new molecules has decreased. To cope with this concern, studies have focused on the exploitation of actinobacteria from unexplored environments and actinobacteria symbionts of plants and animals. In this study, twenty-four actinobacteria strains isolated from workers of Trachymyrmex ants were evaluated for antifungal activity towards a variety of Candida species. Results revealed that seven strains inhibited the tested Candida species. Streptomyces sp. TD025 presented potent and broad spectrum of inhibition of Candida and was selected for the isolation of bioactive molecules. From liquid shake culture of this bacterium, we isolated the rare antimycin urauchimycins A and B. For the first time, these molecules were evaluated for antifungal activity against medically important Candida species. Both antimycins showed antifungal activity, especially urauchimycin B. This compound inhibited the growth of all Candida species tested, with minimum inhibitory concentration values equivalent to the antifungal nystatin. Our results concur with the predictions that the attine ant-microbe symbiosis may be a source of bioactive metabolites for biotechnology and medical applications.