Burkholderia from Fungus Gardens of Fungus-Growing Ants Produces Antifungals That Inhibit the Specialized Parasite Escovopsis.

Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants' fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus converting the plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis. Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia and Paraburkholderia from 50% of the 52 colonies sampled, indicating that members of the family Burkholderiaceae are common inhabitants in the fungus gardens of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1 cm. Genomic assessment of fungus garden-associated Burkholderiaceae indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed that these compounds are responsible for antifungal activities that inhibit Escovopsis but, at equivalent concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of fungus-growing ant colonies. IMPORTANCE Many organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect Leucoagaricus spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, Escovopsis. The ants take multiple approaches, including weeding their fungus gardens to remove Escovopsis spores, as well as harboring Pseudonocardia spp., bacteria that produce antifungals that inhibit Escovopsis. In addition, a genus of bacteria commonly found in fungus gardens, Burkholderia, is known to produce secondary metabolites that inhibit Escovopsis spp. In this study, we isolated Burkholderia spp. from fungus-growing ants, assessed the isolates' ability to inhibit Escovopsis spp., and identified two compounds responsible for inhibition. Our findings suggest that Burkholderia spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite Escovopsis.

Disturbance-diversity relationships of microbial communities change based on growth substrate.

Disturbance events can impact ecological community dynamics. Understanding how communities respond to disturbances and how those responses can vary is a challenge in microbial ecology. In this study, we grew a previously enriched specialized microbial community on either cellulose or glucose as a sole carbon source and subjected them to one of five different disturbance regimes of varying frequencies ranging from low to high. Using 16S rRNA gene amplicon sequencing, we show that the community structure is largely driven by substrate, but disturbance frequency affects community composition and successional dynamics. When grown on cellulose, bacteria in the genera Cellvibrio, Lacunisphaera, and Asticcacaulis are the most abundant microbes. However, Lacunisphaera is only abundant in the lower disturbance frequency treatments, while Asticcacaulis is more abundant in the highest disturbance frequency treatment. When grown on glucose, the most abundant microbes are two Pseudomonas sequence variants and a Cohnella sequence variant that is only abundant in the highest disturbance frequency treatment. Communities grown on cellulose exhibited a greater range of diversity (1.95-7.33 Hill 1 diversity) that peaks at the intermediate disturbance frequency treatment or one disturbance every 3 days. Communities grown on glucose, however, ranged from 1.63 to 5.19 Hill 1 diversity with peak diversity at the greatest disturbance frequency treatment. These results demonstrate that the dynamics of a microbial community can vary depending on substrate and the disturbance frequency and may potentially explain the variety of diversity-disturbance relationships observed in microbial systems.IMPORTANCEA generalizable diversity-disturbance relationship (DDR) of microbial communities remains a contentious topic. Various microbial systems have different DDRs. Rather than finding support or refuting specific DDRs, we investigated the underlying factors that lead to different DDRs. In this study, we measured a cellulose-enriched microbial community's response to a range of disturbance frequencies from high to low, across two different substrates: cellulose and glucose. We demonstrate that the community displays a unimodal DDR when grown on cellulose and a monotonically increasing DDR when grown on glucose. Our findings suggest that the same community can display different DDRs. These results suggest that the range of DDRs we observe across different microbial systems may be due to the nutritional resources microbial communities can access and the interactions between bacteria and their environment.

Disturbance-Diversity Relationships of Microbial Communities Change Based on Growth Substrate.

Disturbance events can impact ecological community dynamics. Understanding how communities respond to disturbances, and how those responses can vary, is a challenge in microbial ecology. In this study, we grew a previously enriched specialized microbial community on either cellulose or glucose as a sole carbon source, and subjected them to one of five different disturbance regimes of varying frequencies ranging from low to high. Using 16S rRNA gene amplicon sequencing, we show that community structure is largely driven by substrate, but disturbance frequency affects community composition and successional dynamics. When grown on cellulose, bacteria in the genera Cellvibrio, Lacunisphaera, and Asticaccacaulis are the most abundant microbes. However, Lacunisphaera is only abundant in the lower disturbance frequency treatments, while Asticaccaulis is more abundant in the highest disturbance frequency treatment. When grown on glucose, the most abundant microbes are two Pseudomonas sequence variants, and a Cohnella sequence variant that is only abundant in the highest disturbance frequency treatment. Communities grown on cellulose exhibited a greater range of diversity (0.67-1.99 Shannon diversity and 1.38-5.25 Inverse Simpson diversity) that peak at the intermediate disturbance frequency treatment, or 1 disturbance every 3 days. Communities grown on glucose, however, ranged from 0.49-1.43 Shannon diversity and 1.37- 3.52 Inverse Simpson with peak diversity at the greatest disturbance frequency treatment. These results demonstrate that the dynamics of a microbial community can vary depending on substrate and the disturbance frequency, and may potentially explain the variety of diversity-disturbance relationships observed in microbial ecosystems.