Nitrogen has a greater influence than phosphorus on the diazotrophic community in two successive crop seasons in Northeast China

Abstract

Fertilizer-induced changes in soil nutrients regulate nitrogen (N) fixation in the terrestrial biosphere, but the influences of N and phosphorus (P) fertilization on the diazotroph communities in successive crop seasons were unclear. In this study, we assessed the effects of N and P (high vs. low doses) on the abundance and structure of N₂-fixation communities after wheat and soybean harvest in a long-term (34 and 35 years) fertilization experiment. In both seasons, long-term N addition significantly decreased the abundance of nifH genes and 16S rDNA; in addition, high doses of N and P fertilizer decreased the richness of diazotrophs, whereas low doses did not. The proportion of the dominant genus, Bradyrhizobium, in the soybean season (86.0%) was higher than that in the wheat season (47.9%). Fertilization decreased diazotroph diversity and the relative abundance of Bradyrhizobium in the wheat season, but had insignificant effects in the soybean season. The addition of N, but not P, significantly changed the communities of both diazotrophs (at the genus level) and rhizobia (at the species level) in the two seasons. Soil pH was positively associated with nifH abundance and diazotrophic richness; soil NO₃^- content was negatively correlated with diazotrophic richness and positively correlated with diversity. Soil pH and NO₃^- content were the two main drivers shaping the soil diazotrophic community. Overall, long-term inorganic N had a greater influence than P on both diazotrophic abundance and community composition, and diazotrophic diversity was more clearly affected by fertilization in the wheat season than in the soybean season.

Figure 1

Absolute abundances of the nifH gene and 16S rDNA in two crop seasons. (A) The absolute abundance of the nifH gene. (B) The absolute abundance of 16S rDNA; (C) The ratio of nitrogen-fixing microorganisms to bacteria.

Figure 2

α diversity indices of nitrogen-fixing microorganisms in soil samples under different fertilizer treatments. (A) Chao index, (B) Shannon index.

Figure 3

Phylogenetic tree and clustering analysis based on nitrogen-fixing microorganisms. (A) Phylogenetic tree displaying the taxonomic information on soil diazotrophic phylotypes. (B,C) show the results of clustering analysis based on nitrogen-fixing microorganisms (at the genus level) in soils of the wheat and soybean seasons, respectively.

Figure 4

Correlation and cluster analysis at the species level. (A) Correlationships between microbial abundance, genera belonging to rhizobia and soil chemical properties. (B,C) show the results of clustering analysis based on rhizobia (at the species level) in soils of the wheat and soybean seasons, respectively. (D) Shows the relative abundance of Bradyrhizobium diazoefficiens in soil samples and their correlation with N fertilizer. W soil samples in the wheat season, S soil samples in the soybean season.

Figure 5

The relationship between the N-fixing community composition and soil chemical properties. (A) Multivariate regression tree (MRT) analysis was performed to interpret the relationship between the nitrogen-fixing community composition and soil chemical properties at the class level. (B) Redundancy analysis (RDA) was performed to assess the relationship between the nitrogen-fixing community composition and soil chemical properties at the genus level. W soil samples in the wheat season, S soil samples in the soybean season.

Figure 6

Experimental design and sample collection.