Fungus Garden Research Articles

Yet more "weeds" in the garden: fungal novelties from nests of leaf-cutting ants.

BackgroundSymbiotic relationships modulate the evolution of living organisms in all levels of biological organization. A notable example of symbiosis is that of attine ants (Attini; Formicidae: Hymenoptera) and their fungal cultivars (Lepiotaceae and Pterulaceae; Agaricales: Basidiomycota). In recent years, this mutualism has emerged as a model system for studying coevolution, speciation, and multitrophic interactions. Ubiquitous in this ant-fungal symbiosis is the “weedy” fungus Escovopsis (Hypocreales: Ascomycota), known only as a mycoparasite of attine fungal gardens. Despite interest in its biology, ecology and molecular phylogeny—noting, especially, the high genetic diversity encountered—which has led to a steady flow of publications over the past decade, only two species of Escovopsis have formally been described.Methods and ResultsWe sampled from fungal gardens and garden waste (middens) of nests of the leaf-cutting ant genus Acromyrmex in a remnant of subtropical Atlantic rainforest in Minas Gerais, Brazil. In culture, distinct morphotypes of Escovopsis sensu lato were recognized. Using both morphological and molecular analyses, three new species of Escovopsis were identified. These are described and illustrated herein—E. lentecrescens, E. microspora, and E. moelleri—together with a re-description of the genus and the type species, E. weberi. The new genus Escovopsioides is erected for a fourth morphotype. We identify, for the first time, a mechanism for horizontal transmission via middens.ConclusionsThe present study makes a start at assigning names and formal descriptions to these specific fungal parasites of attine nests. Based on the results of this exploratory and geographically-restricted survey, we expect there to be many more species of the genus Escovopsis and its relatives associated with nests of both the lower and higher Attini throughout their neotropical range, as suggested in previous studies.

Fungal Garden Making inside Bamboos by a Non-Social Fungus-Growing Beetle

In fungus-growing mutualism, it is indispensable for host animals to establish gardens of the symbiotic fungus as rapidly as possible. How to establish fungal gardens has been well-documented in social fungus-farming insects, whereas poorly documented in non-social fungus-farming insects. Here we report that the non-social, fungus-growing lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) transmits the symbiotic yeast Wickerhamomyces anomalus from the ovipositor-associated mycangium into bamboo internode cavities and disperses the yeast in the cavities to make gardens. Microbial isolation and cryo-scanning electron microscopy observation revealed that W. anomalus was constantly located on the posterior ends of eggs, where larvae came out, and on the inner openings of oviposition holes. Direct observation of oviposition behavior inside internodes revealed that the distal parts of ovipositors showed a peristaltic movement when they were in contact with the posterior ends of eggs. Rearing experiments showed that W. anomalus was spread much more rapidly and widely on culture media and internodes in the presence of the larvae than in the absence. These results suggest that the ovipositors play a critical role in vertical transmission of W. anomalus and that the larvae contribute actively to the garden establishment, providing a novel case of fungal garden founding in non-social insect-fungus mutualism.

Nest architecture, fungus gardens, queen, males and larvae of the fungus-growing ant Mycetagroicus inflatus Brandão & Mayhé-Nunes

All known fungus-growing ants (tribe Attini) are obligately symbiotic with their cultivated fungi. The fungal cultivars of “lower” attine ants are facultative symbionts, capable of living apart from ants, whereas the fungal cultivars of “higher” attine ants, including leaf-cutting genera Atta and Acromyrmex, are highly specialized, obligate symbionts. Since higher attine ants and fungi are derived from lower attine ants and fungi, understanding the evolutionary transition from lower to higher attine agriculture requires understanding the historical sequence of change in both ants and fungi. The biology of the poorly known ant genus Mycetagroicus is of special interest in this regard because it occupies a phylogenetic position intermediate between lower and higher ant agriculture. Here, based on the excavations of four nests in Pará, Brazil, we report the first biological data for the recently described species Mycetagroicus inflatus, including the first descriptions of Mycetagroicus males and larvae. Like M. cerradensis, the only other species in the genus for which nesting biology is known, the garden chambers of M. inflatus are unusually deep and the garden is most likely relocated vertically in rainy and dry seasons. Due to the proximity of nests to the Araguaia River, it is likely that even the uppermost chambers and nest entrances of M. inflatus are submerged during the rainy season. Most remarkably, all three examined colonies of M. inflatus cultivate the same fungal species as their congener, M. cerradensis, over 1,000 km away, raising the possibility of long-term symbiont fidelity spanning speciation events within the genus.

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 etal. 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 etal. (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.

Influência do ambiente no desenvolvimento de colônias iniciais de formigas cortadeiras (Atta sexdens rubropilosa)

Anualmente, as colônias maduras de Atta spp. produzem formigas aladas, machos e fêmeas, que abandonam a colônia na qual foram produzidas para formarem novas colônias e, dessa forma, continuarem a perpetuação da espécie. A fundação de colônias de Atta spp. sempre envolve grande esforço da rainha, única responsável pelo cultivo do fungo e pelos cuidados consigo mesma e com sua prole. Nesse período de fundação ocorre o consumo de grande parte de suas reservas. O presente estudo investigou como o ambiente influencia o desenvolvimento das colônias recém-fundadas de Atta sexdens rubropilosa. Para tanto, foram comparadas colônias de campo coletadas cinco meses após a revoada e colônias mantidas em laboratório desde o voo nupcial. Foram analisados o peso e o tamanho da população (número de operárias, de larvas, de pupas e de ovos), o tamanho das operárias, o volume e peso do jardim de fungo e o esforço para a fundação dos ninhos (teor de lipídeos e peso das rainhas). Comparadas às colônias de campo, as colônias de laboratório apresentaram maiores valores no tamanho e no peso da população, no volume e no peso do jardim de fungo. As medidas das operárias, por classe de tamanho, não variaram entre as colônias de campo e de laboratório. O esforço da fundação dos ninhos foi maior nas colônias de campo, cujas rainhas apresentaram menor porcentagem de lipídeos e menor peso em relação às colônias de laboratório. Esses resultados demonstram que o ambiente exerce forte influência no desenvolvimento das colônias durante a fase de fundação, bem como sobre o status fisiológico das rainhas, sendo, portanto, a sobrevivência, o desenvolvimento e o sucesso da colônia reflexos das condições do ambiente e do esforço da rainha.

Task Performance of Midden Workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae)

Material brought to midden piles of leaf-cutting ants is considered to be hazardous. It is therefore expected that midden workers should not re-enter the colony, to reduce pathogen transmission. Here, we examined whether the midden workers of Atta sexdens rubropilosa remain confined to the waste compartment and, if not, whether they could perform many different behaviors in the absence of specialized nestmates. Eleven subcolonies received either midden workers or non-midden workers in addition to pupae and 50 mL of fungus garden. Survival, fungal and brood care, foraging and waste manipulation were observed daily until subcolonies death. Subcolonies maintained by midden workers died earlier: average survival times were 13.92 and 22.66 days for midden and non-midden workers, respectively. Midden workers cared for the brood and foraged as non-midden workers did but they were not as efficient in caring for the garden. Activities related to waste manipulation were more frequently performed by midden workers. These results show that midden workers are not a behaviorally rigid caste and retain many labor capabilities being able to leave the waste compartment and perform internal activities in the absence of specialized internal workers.

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.

Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.

O jardim de fungo atua como um molde para a construção das câmaras em formigas cortadeiras?

Os ninhos adultos das formigas cortadeiras (gênero Atta e Acromyrmex) são compostos de milhares de câmaras subterrâneas, as quais abrigam o jardim de fungo, lixo e a população desses insetos. Entretanto, como as câmaras são construídas? Para responder essa questão, nós hipotetizamos que o jardim de fungo atua como um molde para a construção das câmaras. Assim, foram utilizadas 20 colônias de 6 meses de idade, divididas em quatro séries experimentais: padrão (quantidade normal de jardim de fungo); metade de jardim de fungo; dobro de jardim de fungo e sem jardim de fungo (Testemunha). As variáveis estudadas foram: parâmetros morfológicos das estruturas (túneis e câmara formada); fluxo das atividades das operárias; volume de solo escavado. Como se esperava, o jardim de fungo atua como um molde para a construção das câmaras em formigas cortadeiras. Os resultados foram: o tratamento sem jardim de fungo não apresentou nenhuma câmara, apenas túneis, em contraposição às demais séries experimentais que apresentaram no mínimo 2 câmaras, com dimensões similares; o fluxo das operárias carregando pellet de solo por minuto durante as 72 horas diferiu estatisticamente entre as séries experimentais e, finalmente, o volume do solo escavado foi resultado da taxa de escavação das operárias, diferindo estatisticamente entre as séries experimentais. Os resultados corroboram a hipótese de que o jardim de fungo atua como um molde para a construção da câmara. A ausência de uma estrutura funcional como uma câmara, quando o jardim de fungo está ausente, comprova a hipótese.

Complete Genome of Serratia sp. Strain FGI 94, a Strain Associated with Leaf-Cutter Ant Fungus Gardens

Serratia sp. strain FGI 94 was isolated from a fungus garden of the leaf-cutter ant Atta colombica. Analysis of its 4.86-Mbp chromosome will help advance our knowledge of symbiotic interactions and plant biomass degradation in this ancient ant-fungus mutualism.

Differential gene expression in Acromyrmex leaf‐cutting ants after challenges with two fungal pathogens

Social insects in general and leaf-cutting ants in particular have increased selection pressures on their innate immune system due to their social lifestyle and monoclonality of the symbiotic fungal cultivar. As this symbiosis is obligate for both parties, prophylactic behavioural defences against infections are expected to increase either ant survival or fungus-garden survival, but also to possibly trade off when specific infections differ in potential danger. We examined the effectiveness of prophylactic behaviours and modulations of innate immune defences by a combination of inoculation bioassays and genome-wide transcriptomic studies (RNA-Seq), using an ant pathogen (Metarhizium brunneum) and a fungus-garden pathogen (Escovopsis weberi) and administering inoculations both directly and indirectly (via the symbiotic partner). Upon detection of pathogen conidia, ant workers responded by increasing both general activity and the frequency of specific defence behaviours (self-grooming, allo-grooming, garden-grooming) independent of the pathogen encountered. This trend was also evident in the patterns of gene expression change. Both direct and indirect (via fungus garden) inoculations with Metarhizium induced a general up-regulation of gene expression, including a number of well-known immune-related genes. In contrast, direct inoculation of the fungus garden by Escovopsis induced an overall down-regulation of ant gene expression, whereas indirect inoculation (via the ants) did not, suggesting that increased activity of ants to remove this fungus-garden pathogen is costly and involves trade-offs with the activation of other physiological pathways.

Complete Genome of Enterobacteriaceae Bacterium Strain FGI 57, a Strain Associated with Leaf-Cutter Ant Fungus Gardens

The Enterobacteriaceae bacterium strain FGI 57 was isolated from a fungus garden of the leaf-cutter ant Atta colombica. Analysis of its single 4.76-Mbp chromosome will shed light on community dynamics and plant biomass degradation in ant fungus gardens.

The fungal symbiont of Acromyrmex leaf-cutting ants expresses the full spectrum of genes to degrade cellulose and other plant cell wall polysaccharides

BackgroundThe fungus gardens of leaf-cutting ants are natural biomass conversion systems that turn fresh plant forage into fungal biomass to feed the farming ants. However, the decomposition potential of the symbiont Leucocoprinus gongylophorus for processing polysaccharides has remained controversial. We therefore used quantifiable DeepSAGE technology to obtain mRNA expression patterns of genes coding for secreted enzymes from top, middle, and bottom sections of a laboratory fungus-garden of Acromyrmex echinatior leaf-cutting ants.ResultsA broad spectrum of biomass-conversion-relevant enzyme genes was found to be expressed in situ: cellulases (GH3, GH5, GH6, GH7, AA9 [formerly GH61]), hemicellulases (GH5, GH10, CE1, GH12, GH74), pectinolytic enzymes (CE8, GH28, GH43, PL1, PL3, PL4), glucoamylase (GH15), α-galactosidase (GH27), and various cutinases, esterases, and lipases. In general, expression of these genes reached maximal values in the bottom section of the garden, particularly for an AA9 lytic polysaccharide monooxygenase and for a GH5 (endocellulase), a GH7 (reducing end-acting cellobiohydrolase), and a GH10 (xylanase), all containing a carbohydrate binding module that specifically binds cellulose (CBM1). Although we did not directly quantify enzyme abundance, the profile of expressed cellulase genes indicates that both hydrolytic and oxidative degradation is taking place.ConclusionsThe fungal symbiont of Acromyrmex leaf-cutting ants can degrade a large range of plant polymers, but the conversion of cellulose, hemicellulose, and part of the pectin occurs primarily towards the end of the decomposition process, i.e. in the bottom section of the fungus garden. These conversions are likely to provide nutrients for the fungus itself rather than for the ants, whose colony growth and reproductive success are limited by proteins obtained from ingesting fungal gongylidia. These specialized hyphal tips are hardly produced in the bottom section of fungus gardens, consistent with the ants discarding old fungal biomass from this part of the garden. The transcripts that we found suggest that actively growing mycelium in the bottom of gardens helps to maintain an optimal water balance to avoid hyphal disintegration, so the ants can ultimately discard healthy rather than decaying and diseased garden material, and to buffer negative effects of varying availability and quality of substrate across the seasons.

Queen lipid content and nest growth in the leaf cutting ant (Atta sexdens rubropilosa) (Hymenoptera: Formicidae)

The relationship between the queens’ lipid content and nest growth (population size, biomass and nest architecture) was studied from founding up to 1 year. Nests aged 3, 4, 5, 6, 9 and 12 months were dug in the field, and their dimensions were measured. The ant nest population and fungus garden was also collected. The sample was taken to the laboratory where we counted the worker population and weighed the biomass (fungus plus offspring) and queens. Queens were separated for the determination of lipids. The lipid content in the bodies of queens decreased in the first months, then stabilized (at 4–6 months) before increasing in months 9 and 12. Nest biomass (symbiotic fungus and offspring) and worker population increased over time. The structural growth of the nests was observed by excavating around them. Initially nests (3 months old) had one chamber at an average depth of 15 cm. By 1 year, the nests had three or four deep chambers, and were about 3–4 m deep. Our study contributes to knowledge of the dynamics of the energy-reserve expenditure by queens during colony founding and colony development for up to 1 year.

Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts

Leaf-cutting ants combine large-scale herbivory with fungus farming to sustain advanced societies. Their stratified colonies are major evolutionary achievements and serious agricultural pests, but the crucial adaptations that allowed this mutualism to become the prime herbivorous component of neotropical ecosystems has remained elusive. Here we show how coevolutionary adaptation of a specific enzyme in the fungal symbiont has helped leaf-cutting ants overcome plant defensive phenolic compounds. We identify nine putative laccase-coding genes in the fungal genome of Leucocoprinus gongylophorus cultivated by the leaf-cutting ant Acromyrmex echinatior. One of these laccases (LgLcc1) is highly expressed in the specialized hyphal tips (gongylidia) that the ants preferentially eat, and we confirm that these ingested laccase molecules pass through the ant guts and remain active when defecated on the leaf pulp that the ants add to their gardens. This accurate deposition ensures that laccase activity is highest where new leaf material enters the fungus garden, but where fungal mycelium is too sparse to produce extracellular enzymes in sufficient quantities to detoxify phenolic compounds. Phylogenetic analysis of LgLcc1 ortholog sequences from symbiotic and free-living fungi revealed significant positive selection in the ancestral lineage that gave rise to the gongylidia-producing symbionts of leaf-cutting ants and their non-leaf-cutting ant sister group. Our results are consistent with fungal preadaptation and subsequent modification of a particular laccase enzyme for the detoxification of secondary plant compounds during the transition to active herbivory in the ancestor of leaf-cutting ants between 8 and 12 Mya.

Termitotrox cupido sp. n. (Coleoptera, Scarabaeidae), a new termitophilous scarab species from the Indo-Chinese subregion, associated with Hypotermes termites

Termitotrox cupido sp. n. is described from Cambodia and represents the first discovery of Termitotrox Reichensperger, 1915 from the Indo-Chinese subregion of the Oriental region. The type series was collected from fungus garden cells of Hypotermes makhamensis Ahmad, 1965 (Isoptera, Termitidae, Macrotermitinae). Hypotermes Holmgren, 1917 was previously an unknown host of Termitotrox species. The new species is readily distinguished from all known congeners by having wing-shaped trichomes on the elytra and is most probably the world’s smallest scarab, at 1.2 mm in length.

Ectosymbionts and immunity in the leaf-cutting ant Acromyrmex subterraneus subterraneus

Associations with symbiotic organisms can serve as a strategy for social insects to resist pathogens. Antibiotics produced by attine ectosymbionts (Actinobacteria) suppress the growth of Escovopsis spp., the specialized parasite of attine fungus gardens. Our objective was to evaluate whether the presence or absence of symbiotic actinobacteria covering the whole ant cuticle is related to differential immunocompetence, respiratory rate and cuticular hydrocarbons (CHs). We evaluated these parameters in three worker groups of Acromyrmex subterraneus subterraneus: External workers (EXT), internal workers with actinobacteria covering the whole body (INB) and internal workers without actinobacteria covering the whole body (INØ). We also eliminated the actinobacteria by antibiotic treatment and examined worker encapsulation response. INB ants showed lower rates of encapsulation and respiration than did the EXT and INØ ants. The lower encapsulation rate did not seem to be a cost imposed by actinomycetes because the elimination of the actinomycetes did not increase the encapsulation rate. Instead, we propose that actinobacteria confer protection to young workers until the maturation of their immune system. Actinobacteria do not seem to change nestmate recognition in these colonies. Although it is known that actinobacteria have a specific action against Escovopsis spp., our studies, along with other independent studies, indicate that actinomycetes may also be important for the individual health of the workers.

Abundance and dynamics of filamentous fungi in the complex ambrosia gardens of the primitively eusocial beetleXyleborinus saxeseniiRatzeburg (Coleoptera: Curculionidae, Scolytinae)

Insect fungus gardens consist of a community of interacting microorganisms that can have either beneficial or detrimental effects to the farmers. In contrast to fungus-farming ants and termites, the fungal communities of ambrosia beetles and the effects of particular fungal species on the farmers are largely unknown. Here, we used a laboratory rearing technique for studying the filamentous fungal garden community of the ambrosia beetle, Xyleborinus saxesenii, which cultivates fungi in tunnels excavated within dead trees. Raffaelea sulfurea and Fusicolla acetilerea were transmitted in spore-carrying organs by gallery founding females and established first in new gardens. Raffaelea sulfurea had positive effects on egg-laying and larval numbers. Over time, four other fungal species emerged in the gardens. Prevalence of one of them, Paecilomyces variotii, correlated negatively with larval numbers and can be harmful to adults by forming biofilms on their bodies. It also comprised the main portion of garden material removed from galleries by adults. Our data suggest that two mutualistic, several commensalistic and one to two pathogenic filamentous fungi are associated with X.saxesenii. Fungal diversity in gardens of ambrosia beetles appears to be much lower than that in gardens of fungus-culturing ants, which seems to result from essential differences in substrates and behaviours.

Fitness consequences of nest infiltration by the mutualist‐exploiter Megalomyrmex adamsae

1. Fungus‐growing ants are obligate mutualists. Their nutrient‐rich fungus garden provides a valuable food store that sustains the ant hosts, but can also attract social parasites.2. The ‘guest ant' Megalomyrmex adamsae Longino parasitises the fungus‐growing Trachymyrmex zeteki Weber queen just after nest founding. The parasitic queen infiltrates the incipient nest, builds a cavity in the fungal garden, and lays eggs that develop into workers and reproductive males and females.3. This study compared young parasitised and non‐parasitised laboratory colonies by measuring garden growth and biomass, and the number of host workers and reproductives. Host queen survival and parasite colony growth were also monitored.4. Parasitised Trachymyrmex colonies had reduced host worker and alate numbers, as well as lower garden biomass, compared with non‐parasitised control colonies, confirming that M. adamsae is a xenobiotic social parasite. Host queen survival was not significantly different between parasitised and control colonies.5. This is the first study that experimentally infects host colonies with a xenobiotic social parasite to measure fitness cost to the host. The natural history of M. adamsae and the fungus‐growing ant mutualism are evaluated in the context of three general predictions of (Bronstein, Ecology Letters, 4, 277–287, 2001a) regarding the cost of mutualism exploiters.

Endophytic fungi increase the processing rate of leaves by leaf‐cutting ants (Atta)

1. Fungal endophytes are microfungi that reside asymptomatically inside of leaf tissues, increasing in density and diversity through time after leaves flush. Previous studies have suggested that the presence of fungal endophytes in the harvest material of leaf‐cutting ants (Atta colombica, Guérin‐Méneville) may negatively affect the ants and their fungal cultivar.2. In the present study, it was tested whether the presence and diversity of fungal endophytes affected the amount of time necessary for leaf‐cutter ants to cut, process, and plant leaf material in their fungal garden. It was found that ants took 30–43% longer to cut, carry, clean, and plant leaf tissue with high relative to low endophyte abundance, and that the ants responded similarly to leaf tissue with high or low endophyte diversity.3. It was further investigated whether the fungal cultivars' colonisation rate was greater on leaf material without fungal endophytes. No difference in the ants' cultivar colonisation rate on leaf tissue with high or low endophyte abundance was observed.