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A powerful mechanism for protection against disease in animals is synergy between metabolites present in the natural microbiota of the host and antimicrobial peptides (AMPs) produced by the host. We studied this method of protection in amphibians in regard to the lethal disease chytridiomycosis, which is caused by Batrachochytrium dendrobatidis (Bd). In this study, we show that the AMPs of Rana muscosa, as well as the metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) from Pseudomonas fluorescens, a bacterial species normally found on the skin of R. muscosa, were inhibitory to the growth of Bd in vitro. When both AMPs and 2,4-DAPG were used in growth inhibition assays, they worked synergistically to inhibit the growth of Bd. This synergy resulted in reduced minimum concentrations necessary for inhibition by either 2,4-DAPG or AMPs. This inhibitory concentration of AMPs did not inhibit the growth of a P. fluorescens strain that produced 2,4-DAPG in vitro, although its growth was inhibited at higher peptide concentrations. These data suggest that the AMPs secreted onto frog skin and the metabolites secreted by the resident beneficial bacteria may work synergistically to enhance protection against Bd infection on amphibian skin. These results may aid conservation efforts to augment amphibian skins' resistance to chytridiomycosis by introducing anti-Bd bacterial species that work synergistically with amphibian AMPs.

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions. IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

For decades, Amphibians have been globally threatened by the still expanding infectious disease, chytridiomycosis. Madagascar is an amphibian biodiversity hotspot where Batrachochytrium dendrobatidis ( Bd ) has only recently been detected. While no Bd -associated population declines have been reported, the risk of declines is high when invasive virulent lineages become involved. Cutaneous bacteria contribute to host innate immunity by providing defense against pathogens for numerous animals, including amphibians. Little is known, however, about the cutaneous bacterial residents of Malagasy amphibians and the functional capacity they have against Bd . We cultured 3179 skin bacterial isolates from over 90 frog species across Madagascar, identified them via Sanger sequencing of approximately 700 bp of the 16S rRNA gene, and characterized their functional capacity against Bd . A subset of isolates was also tested against multiple Bd genotypes. In addition, we applied the concept of herd immunity to estimate Bd -associated risk for amphibian communities across Madagascar based on bacterial antifungal activity. We found that multiple bacterial isolates (39% of all isolates) cultured from the skin of Malagasy frogs were able to inhibit Bd . Mean inhibition was weakly correlated with bacterial phylogeny, and certain taxonomic groups appear to have a high proportion of inhibitory isolates, such as the Enterobacteriaceae, Pseudomonadaceae, and Xanthamonadaceae (84, 80, and 75% respectively). Functional capacity of bacteria against Bd varied among Bd genotypes; however, there were some bacteria that showed broad spectrum inhibition against all tested Bd genotypes, suggesting that these bacteria would be good candidates for probiotic therapies. We estimated Bd -associated risk for sampled amphibian communities based on the concept of herd immunity. Multiple amphibian communities, including those in the amphibian diversity hotspots, Andasibe and Ranomafana, were estimated to be below the 80% herd immunity threshold, suggesting they may be at higher risk to chytridiomycosis if a lethal Bd genotype emerges in Madagascar. While this predictive approach rests on multiple assumptions, and incorporates only one component of hosts' defense against Bd , their culturable cutaneous bacterial defense, it can serve as a foundation for continued research on Bd -associated risk for the endemic frogs of Madagascar.

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