Phyllosphere antibiotic resistome in a natural primary vegetation across a successional sequence after glacier retreat

The spread of antibiotic-resistance genes (ARGs) has posed a significant threat to human health over the past decades. Despite the fact that the phyllosphere represents a crucial pool of microorganisms, little is known about the profile and drivers of ARGs in less human interference natural habitats. In order to minimize the influence of environmental factors, here we collected leaf samples from the early-, middle- and late-successional stages across a primary vegetation successional sequence within 2 km, to investigate how the phyllosphere ARGs develop in natural habitats. Phyllosphere ARGs were determined using high-throughput quantitative PCR. Bacterial community and leaf nutrient content were also measured to assess their contribution to the phyllosphere ARGs. A total of 151 unique ARGs were identified, covering almost all recognized major antibiotic classes. We further found that there was some stochastic and a core set of the phyllosphere ARGs during the plant community succession process, due to the fluctuant phyllosphere habitat and specific selection effect of plant individuals. The ARG abundance significantly decreased due to the reduction of the phyllosphere bacterial diversity, community complexity, and leaf nutrient content during the plant community succession process. While the closer links between soil and fallen leaf resulted in a higher ARG abundance in leaf litter than in fresh leaf. In summary, our study reveals that the phyllosphere harbors a broad spectrum of ARGs in the natural environment. These phyllosphere ARGs are driven by various environmental factors, including the plant community composition, host leaf properties, and the phyllosphere microbiome.