Different contributions of rare microbes to driving soil nitrogen cycles in acidic soils under manure fertilization

Organic manure can substantially improve soil fertility and influence soil microbes. Despite the pivotal role of Ncycling microbes in agro-ecosystems, little is known about the importance of rare microbes to N cycles in acidic soils under manure fertilization. Here, we conducted a 11 -year field experiment in which chemical N fertilizers were substituted with swine manure at different doses in an acidic soil. We investigated soil biological N fixation (BNF), potential nitrification rate (PNR), denitrifying enzyme activity (DEA), and associated microbial communities (diazotrophs, ammonia oxidizing bacteria (AOB), ammonia oxidizing archaea (AOA), and nirK- and nirS-type denitrifiers). With the increase of manure dose, BNF, PNR, DEA and abundances of soil N-cycling functional genes significantly increased. Manure application changed whole and subgroup (abundant, intermediate, and rare taxa) community structures of these N-cycling microbes and increased Shannon indexes of AOA, AOB and nirK-type denitrifiers but not diazotrophs and nirS-type denitrifiers. The community structures and Shannon variations of rare AOA and AOB subgroups highly contributed to PNR and subgroup community structures of rare nirK- and nirS-type denitrifiers were significantly related to DEA, but rare diazotrophs did not contribute to BNF. In the co-occurrence networks of AOA, AOB, nirK- and nirS-type denitrifiers, most keystone species were rare taxa, and the positive edge numbers belonging to rare taxa were positively related to PNR and DEA. Manure-derived microbial species provided the very low contributions to community dissimilarities of these soil N-cycling microbes. Our findings suggested that rare microbes made surprisingly important contributions to soil nitrification and denitrification rather than N-fixation in manured acidic soils.