Asymbiotic Nitrogen Fixation in the Phyllosphere of Urban Green Spaces.

Biological nitrogen fixation (BNF) is an important source of nitrogen in ecosystems. Compared to symbiotic nitrogen-fixing microorganisms, free-living diazotrophic bacteria have a broader distribution and greater diversity, demonstrating greater potential for application. Leaf surfaces constitute one of the largest microbial reservoirs on Earth, harboring a variety of free-living diazotrophic bacteria, contributing significantly to plant N acquisition and growth. The distribution patterns, abundance, diversity, and the environmental variables affecting the asymbiotic nitrogen fixation (ANF) rates of free-living diazotrophic bacteria of non-leguminous plants in urban green spaces were investigated using high-throughput sequencing of nifH gene amplicons and the acetylene reduction method. Both green space type and plant species significantly impact ANF rates and nifH gene abundance in the phyllosphere, with green space type having a more pronounced effect. Leaf metal elements iron (Fe), molybdenum (Mo), and the free-living diazotrophic bacteria of the genus Skermanella collectively influence the ANF rates in the phyllosphere of urban green spaces. Linear regression analysis revealed that metal elements Fe, Mo, and potassium (K) in the leaves were significantly positive correlated with the diversity of the free-living diazotrophic bacteria and the abundance of the N-fixing gene nifH. The alpha diversity and symbiotic network structure of the free-living diazotrophic bacterial community in the phyllosphere indicated a significant negative correlation between human disturbance and environmental perturbation and the biodiversity and network complexity of these bacteria. This study provides a crucial foundation for understanding the nitrogen-fixing functions of microbes in urban ecosystems and their contributions to the nitrogen cycle.