Structure and assembly cues for rhizospheric nirK- and nirS-type denitrifier communities in long-term fertilized soils

The stimulatory effect of plants on soil denitrification activity has been widely reported, and root-derived carbon (C) is considered a factor contributing to enhanced denitrification activity in the rhizosphere. However, the mechanisms through which root-derived C shapes the rhizospheric denitrifying community structure remains unclear, especially under different soil fertility levels. Here, DNA-based stable isotope probing (DNA-SIP) and Illumina MiSeq sequencing were employed to characterize root-associated denitrifier communities containing nitrite reductase genes (nirS and nirK) in the rhizosphere of wheat grown in soils with three distinct long-term (32-year) fertilization regimes. Fertilization showed a significant impact on the composition of denitrifying communities that actively utilized photosynthetically fixed C in the wheat rhizosphere. In soils amended with inorganic fertilizers, wheat root-derived 13C was mainly assimilated by nirS-type denitrifiers affiliated to Alcaligenaceae and nirK-type denitrifiers affiliated to Phyllobacteriaceae. In contrast, organic fertilization resulted in larger diversity of 13C-labeled denitrifier communities where nirS-type denitrifiers such as Rhodobacteraceae and unclassified Burkholderiales and some unknown nirK-type denitrifiers were more abundant. The nirS-type denitrifier community was found to be more sensitive to the rhizosphere effect than the nirK-type community. Approximately 31% of the 13C-labeled nirS-type denitrifiers were more abundant in the rhizosphere than in the bulk soil, but only 2% of the 13C-labeled nirK-type denitrifiers showed increased abundance in the rhizosphere. The results of this study present direct evidence that root exudates can act as inducible C sources for heterotrophic denitrifying bacteria, but this induction pattern differs between nirS- and nirK-type communities and is dependent on soil fertility level.