Urban Pollution Shapes the Endophytic Microbiome of Urban Trees: Taxonomic and Functional Insights from Fraxinus excelsior, Tilia tomentosa, and Pinus nigra in Plovdiv, Bulgaria ^†

Urbanization imposes significant environmental pressures on plant microbiomes through pollution, habitat restriction, and microclimate changes. This study investigated the diversity, composition, and functional potential of endophytic bacterial communities inhabiting three urban tree species—Tilia tomentosa, Fraxinus excelsior, and Pinus nigra—sampled from four locations in Plovdiv, Bulgaria. Plants are exposed to different environmental pollutants. High-throughput 16S rRNA gene sequencing and alpha and beta diversity indices reveal that tree host species and urban microenvironmental conditions strongly shape microbial community structure. Fraxinus excelsior and Tilia tomentosa harbor more diverse and functionally enriched endophytic microbiomes compared to Pinus nigra, which has significantly higher relative abundance of beneficial genera such as Microvirga, Brevundimonas, and Arthrobacter. In contrast, Pinus nigra hosts specialized taxa such as Novosphingobium, known for their pollutant-degrading abilities. Beta diversity and NMDS analyses confirmed species-specific clustering, while LEfSe (Linear discriminant analysis Effect Size) and metagenomeSeq analyses identified microbial biomarkers and differentially abundant taxa associated with nitrogen cycling, xenobiotic degradation, and stress reduction. Functional profiling predicted the enrichment of pathways involved in denitrification, methanotrophy, and hydrocarbon degradation, particularly in broadleaf species and highly urbanized plots. These results suggest that urban pollutants exert selective pressure on endophytic assemblages, leading to the enrichment of stress-resilient and metabolically versatile taxa. The functional capacity of these microbiomes likely contributes to host tree adaptation, health, and ecological performance in polluted urban environments. Overall, this study highlights the pivotal role of endophytic microbiomes as both indicators and mediators of urban environmental stress and underscores their potential utility in enhancing urban tree resilience and supporting sustainable green infrastructure through microbiome-informed management strategies.