From the leaf to the gut and back again: the fate and influence of phyllosphere bacteria in a gnotobiotic Arabidopsis – Pieris brassicae system

Background: The leaf surface, or phyllosphere, hosts abundant and diverse bacterial communities that interact with both the host plant and herbivorous insects, yet their collective influence on plant–insect interactions remains poorly investigated. We established a gnotobiotic insect-plant system combining Arabidopsis thaliana and Pieris brassicae larvae. Using defined synthetic phyllosphere communities (SynComs) of increasing richness (5, 10, or 20 members), we investigated how phyllosphere bacteria influence herbivore performance, plant defence responses, and bacterial colonisation of both leaves and the insect gut. Results: While larval weight tended to decrease with increasing community richness, only the most diverse SynCom (20 members) caused significant weight reductions without affecting survival. Likewise, plants harbouring the most diverse community showed enhanced jasmonic acid (JA) levels during feeding, whereas salicylic acid (SA) remained unchanged, suggesting the specific induction of JA-associated defences. Compared to plants experiencing no herbivory, feeding strongly reshaped bacterial colonisation on leaves, increasing total bacterial loads about fourfold and driving dominance of Pantoea eucalypti 299R as shown by 16S rRNA gene amplicon sequencing. Larvae acquired a distinct subset of bacteria, primarily recruited from the genera Methylobacterium , Microbacterium , Williamsia , and Curtobacterium . Conclusions: Together, these findings suggest that resident phyllosphere bacteria modulate plant defences and thereby affect herbivore performance, while herbivory restructures the leaf microbiota and bacterial filtering occurs during passage through the insect gut.