**Background:** The microbiota of liverworts provides an interesting model for plant symbioses, but their microbiome assembly is not yet understood. This study assessed factors shaping microbial communities associated with Riccia temporary agricultural crusts in harvested fields by investigating bacterial, fungal, and archaeal communities in thalli and adhering soil from different field sites in Styria and Burgenland, Austria, using qPCR, amplicon sequencing, and advanced microscopy.
**Methods:** Liverwort samples (Riccia glauca and Riccia bifurca) were collected from corn fields and pumpkin fields at six locations in Austria. From each location, six thalli were collected. Thalli and adhering soil were separated, washed, and DNA extracted. Quantitative real-time PCR quantified prokaryotic 16S rRNA, archaeal 16S rRNA, methylobacterial mxaF, and fungal ITS gene copies. Amplicon sequencing targeted the V4 region of prokaryotic 16S rRNA and fungal ITS1 region on an Illumina MiSeq platform. Bioinformatic analysis used QIIME2 with DADA2, and taxonomic classification against SILVA v132 and UNITE databases. Fluorescence in situ hybridization (FISH) with confocal laser scanning microscopy visualized bacterial colonization. SourceTracker2 estimated soil-origin proportions. LEfSe identified enriched taxa. FUNGuild predicted fungal trophic modes. Cultivation on King's B medium isolated bacteria, identified by 16S Sanger sequencing.
**Key Results:** Riccia thalli harbored very high microbial abundances: 10^10 16S rRNA gene copies/g thallus for bacteria, and 10^8 ITS copies/g for fungi. Each thallus contained approximately 1000 prokaryotic and fungal ASVs. Prokaryotic richness was lower in thalli (observed ASVs: 884; Shannon index: 5.72) vs. soil (observed ASVs: 1327; Shannon index: 6.51; P<0.001). Fungal richness was also lower in thalli (observed ASVs: 132; Shannon index: 3.47) vs. soil (observed ASVs: 192; Shannon index: 3.90; P<0.001). Field type did not affect prokaryotic diversity in thalli (P_adjusted=0.798), but fungal diversity differed: corn field thalli had higher fungal diversity (Shannon: 3.80) vs. pumpkin field thalli (Shannon: 3.31; P_adjusted=0.008). Adonis analysis showed microhabitat explained 14.9% of prokaryotic community variation, while field type explained only 4.1%. For fungi, field type explained 34.1% of variation, and microhabitat explained 12.8% (all P<0.001). Mantel tests revealed fungal communities in thalli were highly correlated with soil (r=0.476, P<0.001), while prokaryotic communities showed weak correlation (r=0.182, P=0.021). Methylobacterial abundance was higher in thalli vs. soil (P<0.001), and higher in pumpkin field thalli (1.8×10^7 to 1.7×10^8 mxaF copies/g) vs. corn field thalli (1.1×10^7 to 1.5×10^5 copies/g). A core microbiome of 38 bacterial ASVs (19.9% of total reads) was identified, dominated by Alphaproteobacteria (e.g., Sphingomonas, Rhizobium) and Gammaproteobacteria (e.g., Rhizobacter, Massilia), plus 5 Oxyphotobacteria ASVs (9.4% of reads). Only 13 fungal core ASVs were found. SourceTracker estimated 71.2% of fungal community in thalli originated from soil, vs. 30.4% for bacteria. LEfSe identified 510 bacterial ASVs enriched in thalli vs. soil, including beneficial genera (Rhizobium, Bradyrhizobium, Flavobacterium, Sphingomonas, Herbaspirillus, Methylobacterium) and pathogenic ASVs closely related to Pseudomonas viridiflava, Ralstonia syzygii, and Xanthomonas campestris. Among 79 enriched fungal ASVs, pathogenic genera included Alternaria, Psathyrella, and Plectosphaerella. Cultivation yielded isolates of Pseudomonas and Xanthomonas, including Xanthomonas translucens (13 isolates) and Pseudomonas asplenii. FISH-CLSM confirmed Alphaproteobacteria and Gammaproteobacteria colonization on thallus surfaces and spores, suggesting vertical transmission.
**Clinical Implications:** This study demonstrates that temporary agricultural biocrusts formed by Riccia liverworts are important reservoirs for microbial biodiversity in post-harvest fields. The stable, host-specific bacterial community, particularly the enrichment of methylobacteria and cyanobacteria, suggests symbiotic interactions that may support liverwort fitness and nutrient cycling. The finding that fungal communities are strongly shaped by crop cultivation history indicates that agricultural practices indirectly influence microbiome assembly. Critically, the enrichment of known bacterial and fungal phytopathogens (e.g., Xanthomonas, Alternaria) in liverwort thalli points to an undescribed role of liverworts as potential pathogen reservoirs that could transmit diseases to subsequent crops. This has implications for agricultural management, suggesting that post-harvest biocrusts should be considered in crop rotation and disease management strategies. The study also provides a foundation for understanding plant microbiome evolution in ancient land plants.