The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis ( Bd ) and Batrachochytrium salamandrivorans ( Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd , with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded ( n = 177) in Bsal , most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal . Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

(1) Background: Microbial communities in terrestrial, calcifying high-alkaline springs are not well understood. In this study, we investigate the structure and composition of microbial mats in ultrabasic (pH 10–12) serpentinite springs of the Voltri Massif (Italy). (2) Methods: Along with analysis of chemical and mineralogical parameters, environmental DNA was extracted and subjected to analysis of microbial communities based upon next-generation sequencing. (3) Results: Mineral precipitation and microbialite formation occurred, along with mat formation. Analysis of the serpentinite spring microbial community, based on Illumina sequencing of 16S rRNA amplicons, point to the relevance of alkaliphilic cyanobacteria, colonizing carbonate buildups. Cyanobacterial groups accounted for up to 45% of all retrieved sequences; 3–4 taxa were dominant, belonging to the filamentous groups of Leptolyngbyaceae, Oscillatoriales, and Pseudanabaenaceae. The cyanobacterial community found at these sites is clearly distinct from creek water sediment, highlighting their specific adaptation to these environments.

Aspergillus fumigatus is the leading cause of the fungal invasive disease called aspergillosis, which is associated with a high mortality rate that can reach 50% in some groups of immunocompromised individuals. The increasing prevalence of azole-resistant A. fumigatus isolates, both in clinical settings and the environment, highlights the importance of discovering new fungal virulence factors that can potentially become targets for novel antifungals. Nitronate monooxygenases (Nmos) represent potential targets for antifungal compounds as no orthologs of those enzymes are present in humans. Nmos catalyse the denitrification of nitroalkanes, thereby detoxifying these mediators of nitro-oxidative stress, and therefore we tested whether Nmos provide protection for A. fumigatus against host-imposed stresses at sites of infection. The results of inhibition zone assays indicated that Nmo2 and Nmo5 are not essential for the oxidative stress resistance of A. fumigatus in vitro. In addition, the resazurin-based metabolic activity assay revealed that the growth of mutants lacking the nmo2 or nmo5 genes was only slightly reduced in the presence of 0.05 mM peroxynitrite. Nevertheless, both Nmo2 and Nmo5 were shown to contribute to defense against murine bone marrow-derived macrophages, and this was no longer observed when NADPH oxidase, the main generator of reactive oxygen species during infection, was inhibited in macrophages. Furthermore, we revealed that Nnmos promote the virulence of the fungus in the Galleria mellonella model of infection. Both nmo2 and nmo5 knock-out strains were less virulent than the wild-type control as recorded 72 h post-infection. Our results indicate that Nmos play a role in the virulence of A. fumigatus.

Downy mildew (DM) diseases are caused by destructive obligate pathogens with limited control options, posing a significant threat to global agriculture. RNA interference (RNAi) has emerged as a promising, environmentally sustainable strategy for disease management. We evaluated the efficacy of dsRNA‐mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana , pea and lettuce: Hyaloperonospora arabidopsidis ( Hpa ), Peronospora viciae f. sp. pisi ( Pvp ) and Bremia lactucae ( Bl ), respectively. Conserved genes, cellulose synthase 3 ( CesA3 ) and beta‐tubulin ( BTUB ), were targeted. Silencing these genes significantly impaired spore germination and infection across species and reduced gene expression correlated with suppressed sporulation, confirming silencing efficacy. We tested dsRNAs from chemical synthesis, in vitro transcription, and Escherichia coli expression. Uptake and silencing efficiency varied with dsRNA length and concentration. In Hpa , short dsRNAs (21–25 bp) produced a variable spore germination rate, with 25 bp dsRNA causing a 247.10% increase, whereas longer dsRNAs (≥ 30 bp) completely inhibited germination. Similarly, in Pvp , dsRNAs of 21–25 bp resulted in a 73.05%–77.46% germination rate, while 30–75 bp dsRNAs abolished germination. Confocal microscopy using Cy‐5‐labelled short‐synthesised dsRNA (SS‐dsRNA) confirmed uptake by spores. Sequence specificity influenced efficacy, highlighting the need for precise target design. Multiplexed RNAi impacted silencing synergistically, further reducing germination and sporulation in Hpa . Importantly, SS‐dsRNA‐mediated silencing was durable, with reduced gene expression sustained at 4, 7, 10 and 11 days post‐inoculation. Taken together, our findings demonstrate the potential of dsRNA‐mediated gene silencing as a precise, sustainable tool for managing DM pathogens in multiple crop species.

Downy mildew (DMs) dieases are caused by destructive obligate pathogens with limited control options, posing a significant threat to global agriculture. RNA interference (RNAi) has emerged as a promising, environmentally sustainable strategy for disease management. In this study, we evaluated the efficacy of dsRNA-mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana , pea and lettuce DM pathogens, Hyaloperonospora arabidopsidis ( Hpa ), Peronospora viciae f. sp. pisi ( Pvp ) and Bremia lactucae ( Bl ), respectively. We specifically targeted the cellulose synthase 3 ( CesA3) and the beta tubulin (BTUB) genes. Silencing CesA3 impaired spore germination and infection across multiple species, while BTUB silencing reinforced the potential of dsRNA-mediated inhibition. Reduction in gene expression levels correlated well with the sporulation assays confirming the effectiveness of dsRNA-mediated gene silencing. We used dsRNAs that were chemically synthesized, in vitro transcribed (IVT) or produced in E. coli . We found that the length and concentration of these dsRNAs significantly affected uptake efficiency, spore germination, and sporulation, with higher concentrations enhancing inhibitory effects. Confocal microscopy using Cy-5-labelled short-synthesized dsRNA (SS-dsRNA) provided direct evidence of spore uptake, confirming the potential of SS-dsRNA for pathogen control. However, species-specific sequence variations influenced dsRNA efficacy, underscoring the importance of target sequence design. Multiplexed RNAi impacted silencing synergisticly, further reducing germination and sporulation in Hpa . Additionally, we demonstrated that SS-dsRNA-mediated gene silencing is sustained over time, with a significant reduction in gene expression level at 4, 7, 10 and 11dpi. This indicates the durability and efficacy of this approach. Taken together, these findings demonstrate the potential of dsRNA-mediated gene silencing as a precision tool for managing DM pathogens.