Effects of long-term fertilization on nifH gene diversity in agricultural black soil

Black soil is a precious resource: it is an important grain-base commodity and a highly fertile soil. The long-term use of chemical and organic fertilisers has affected the microbial community and agricultural sustainability of black soil. We used nitrogenase reductase (nifH) as a biomarker of soil-borne nitrogen-fixing microbes to study the effects of long-term fertilisation on a field. Fertilisation started in 1979 and consists of the following treatments; organic, chemical, chemical and organic fertiliser combination, and no fertiliser (control treatment). The nifH gene pools from each fertilisation treatment were amplified and constructed into four clone libraries. Restriction fragment length polymorphisms (RFLP) analysis was then performed identifying 87 nifH gene types amongst the nitrogen-fixing microbial community. This analysis showed that the chemical and organic fertiliser combination resulted in a notable increase in nifH phylotypes and simultaneously intensified the differences among the unique phylotype quantities. The phylogenetic distribution of the clones based on nifH sequences from the four treatments was similar and sequences aligned with various common soils taxonomic groups, including Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes and Cyanobacteria. The impact of mixed fertilisers on the nifH genotypes was complex and resulted in the selection of unique microbial flora. This study also demonstrated that the addition of organic fertiliser created better environmental conditions for the composition of the nifH pool. Thus, the application of organic fertilisers can greatly increase the types of nitrogen-fixing microorganisms present and is conducive to the development of a rich variety and evolution of nitrogen fixation genes.   Key words: nifH, long-term fertiliser, diversity.