Bennet Rohan Fernando Devasahayam

Background: Barley ( Hordeum vulgare L.) provides a suitable model for studying domestication-driven plant-microbiome interactions. Although wild, landrace, and modern genotypes host distinct rhizosphere communities, the extent to which roots and microbes reciprocally influence each other remains unclear. Here, we applied an integrated multi-omics approach combining long-read metagenomics, root transcriptomics, and plant genomics to understand genotype-specific host-microbiome coordination. Results: Oxford Nanopore whole metagenome sequencing (WMS) revealed genotype-associated shifts in rhizosphere communities across seasons. Functional profiling showed a conserved metabolic backbone including amino acid metabolism, energy production, and secondary metabolite biosynthesis, alongside genotype-dependent variation in carbohydrate metabolism and transport-associated pathways. Genome-resolved analysis through metagenome-assembled genomes (MAGs) further detailed the taxonomic and functional architecture of key rhizosphere lineages. Root transcriptome profiling identified extensive differential expression associated with microbial perception, signaling, defense, and metabolic processes. Integration of host and microbiome data revealed coordinated molecular patterns, indicating that barley genotypes are associated with distinct microbial assemblages and corresponding transcriptional responses. Conclusions: These findings indicate that domestication has shaped coordinated associations between barley genotypes and their rhizosphere microbiomes, reflected in both microbial community composition and host transcriptional regulation. This work provides new insights into the evolutionary tuning of plant-microbiome relationships and highlights opportunities for microbiome-informed strategies in barley improvement.

Microbial interactions in agricultural ecosystems are chemically dynamic, with significant implications for ecological balance and crop protection. As synthetic fungicides face increasing regulatory and resistance challenges, applying microbial biological control agents (MBCAs) presents a potential alternative crop-protection strategy. However, the chemical and toxicological consequences of such applications remain poorly understood. Using the model ascomycete Aspergillus nidulans , the response to confrontations with twelve microbial partners, including bacterial and fungal MBCAs, plant pathogens, and phylloplane isolates was studied. Dual-culture assays revealed distinct interaction patterns, and transcriptome profiling showed confrontation-specific activation of secondary metabolite biosynthetic gene clusters (SMBGCs), with up to 50 SMBGCs differentially expressed in A. nidulans . Complementary untargeted LC-MS/MS identified hundreds of unique secondary metabolites (SMs), including compounds structurally resembling synthetic fungicides, including azoles and piperidines. Notably, cytotoxicity assays using human HEK-293 and HCT-116 cell lines revealed that SM mixtures from confrontations of A. nidulans with Bacillus subtilis or Trichothecium roseum exhibited significant toxicity, with IC₅₀ values as low as 52 µg/mL. These findings demonstrate that microbial confrontations can trigger the production of diverse and specific sets of SMs, including compounds representing potential human health risks. This study underscores the need for confrontation-informed toxicological assessments in MBCA regulation and highlights the importance of developing safer biocontrol strategies in agriculture. IMPORTANCE: This study shows fundamental changes at the transcriptome and metabolome level in the ubiquitous fungus Aspergillus nidulans confronting various microorganisms, including microbial biological control agents. Strong modulations of transcript abundances of genes belonging to secondary metabolism gene clusters correlated with the formation of a vast array of novel secondary metabolites. Compounds formed in some confrontations were toxic to human cells, questioning the consumer safety of applying microbial biological control agents.

Microbial biological control agents are increasingly used as an alternative to synthetic pesticides. The application of these microorganisms massively affects all members of plant‐colonising microbial communities, including pathogenic fungi. In the majority of cases, the resulting competition for ecological niches is decided by the toxicity of microbial secondary metabolites (SMs) formed. In this study, we devised confrontation experiments employing the fungal maize pathogen Colletotrichum graminicola and antagonistic partners, that is the biocontrol bacterium Bacillus amyloliquefaciens and the ubiquitous ascomycete Aspergillus nidulans . Transcriptome studies uncovered strong de‐regulation of the vast majority of the C. graminicola secondary metabolite biosynthetic gene clusters (SMBGCs), with 69% and 86% of these clusters de‐regulated at confrontation sites with B. amyloliquefaciens or A. nidulans , respectively. In the biocontrol bacterium and in A. nidulans confronting the maize pathogen, 100% and 74% of the SMBGCs were transcriptionally de‐regulated, respectively. Correspondingly, non‐targeted high‐resolution LC–MS/MS revealed a large repertoire of 1738 and 1466 novel features formed in the fungus–bacterium and fungus–fungus confrontation, respectively. Surprisingly, several of these belong to chemical classes with lead structures of synthetic fungicides.