Effects of Continuous Nitrogen Fertilizer Application on the Diversity and Composition of Rhizosphere Soil Bacteria

Abstract

Little has been reported on the effects of long-term fertilization on rhizosphere soil microbial diversity. Here, we investigated the effects of long-term continuous nitrogen (N) fertilization on the diversity and composition of soil bacteria using data from a 10-year field experiment with five N application rates (0, 120, 180, 240, and 360 kg N hm^-2). The results revealed varying degrees of reduction in the numbers of bacterial operational taxonomic units (OTUs) in response to the different N application rates. The highest wheat yield and number of proprietary bacterial OTUs were found in the N input of 180 kg N hm^-2. In terms of average relative richness, the top seven phyla of soil bacteria in the rhizosphere of wheat after long-term nitrogen application were Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Bacteroidetes, Gemmatimonadetes, and Patescibacteria. Among these, Proteobacteria and Gemmatimonadetes were found to be unaffected by the nitrogen fertilizer and soil environmental factors (pH, C/N ratio, and NO₃ ^- concentration), whereas Acidobacteria and Actinobacteria showed significant positive and negative correlations, respectively, with soil pH. The richness of Actinobacteria significantly increased in the N₁₈₀ treatment. Patescibacteria and Bacteroidetes showed significant positive correlations with soil NO₃ ^- and wheat yield, and the average relative richness of these two phyla was high under long-term application of the N₁₈₀ treatment. These findings indicate that the relative richness of Patescibacteria and Bacteroidetes can affect wheat yield. In conclusion, the results of our 10-year field experiments clearly show that long-term N fertilization can significantly affect most of the dominant soil bacterial species via changing the soil pH. The richness of Actinobacteria can serve as an indicator of a decreased soil pH caused by long-term N fertilization.

Keywords: average relative richness; long-term N application; soil bacterial; soil pH; wheat yield.

FIGURE 1

Effects of continuous application of different amounts of nitrogen fertilizer on wheat yield.

FIGURE 2

Rhizosphere soil microbial community responses to different long-term nitrogen fertilizer applications. N₀-1, N₀-5, and N₀-10 represent the first, fifth, and 10th year of the 0 kg N hm^–2 treatment, respectively. A similar notation has been used for the other fertilizer treatments.

FIGURE 3

Proportions of the dominant bacterial phyla in different treatment samples. N₀-1, N₀-5, and N₀-10 represent the first, fifth, and 10th year of the 0 kg N hm^–2 treatment, respectively. A similar notation has been used for the other fertilizer treatments. In the relation diagram between the Circos sample and species, the small semicircle (left half circle) represents the species composition in the sample. The color of the outer ribbon represents the group from which it comes. The color of the inner ribbon represents the species, and the length represents the relative richness of the species in the corresponding sample. The large semicircle (right half circle) indicates the distribution proportion of species in the different samples at the taxonomic level. The color of the outer ribbon represents the species, the color of the inner ribbon represents the different groups, and the length represents the distribution proportion of the sample in a certain species.

FIGURE 4

Correlations between the different long-term nitrogen applications and changes in the dominant phyla of the soil bacteria. N₀-1, N₀-5, and N₀-10 represent the first, fifth, and 10th year of the 0 kg N hm^–2 treatment, respectively. A similar notation has been used for the other fertilizer treatments. Y represents the species names at the taxonomic level, X represents the average relative richness of the species in different groups, and the columns of different colors represent different groups. On the far right is the P-value: *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; ***P ≤ 0.001.

FIGURE 5

Correlations between dominant phyla of soil bacteria and environmental factors under different long-term nitrogen applications. N₀-1, N₀-5, and N₀-10 represent the first, fifth and tenth year of the 0 kg N hm⁻² treatment, respectively. A similar notation has been used for the other fertilizer treatments. Labels (A–C) represent the first, fifth and tenth year of fertilizer treatments respectively. The points in the figure with different colors or shapes represent the sample groups under different environments or conditions; The red arrows represent quantitative environmental factors, and the length of the environmental factor arrows can represent the degree of environmental factors’ influence on the species data (explanatory quantity). The included angle between arrows of environmental factors represents positive and negative correlation (acute angle: positive correlation; obtuse angle: negative correlation; right angle: no correlation); From the sample point to the arrows of the quantitative environmental factors, the distance between the projection point and the origin represents the relative influence of the environmental factors on the distribution of the sample community, and the direction of the points and arrows represents the positive and negative correlation.

FIGURE 6

Relationships between the dominant soil bacteria and environmental factors. N₀-1, N₀-5, and N₀-10 represent the first, fifth, and 10th year of the 0 kg N hm^–2 treatment, respectively. A similar notation has been used for the other fertilizer treatments. X and Y are the environmental factors and species, respectively, and the correlation R-value and P-value are obtained by calculation. The R-value is shown in different colors in the figure. If the P-value is < 0.05, it is marked with an asterisk. The legend on the right is the color interval of the different R-values. Cluster trees of the species and environmental factors are presented on the left and upper parts. *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; ***P ≤ 0.001.

FIGURE 7

Correlations among the yield, nitrogen application, and dominant soil bacteria. N₀-1, N₀-5, and N₀-10 represent the first, fifth, and 10th year of the 0 kg N hm^–2 treatment, respectively. A similar notation has been used for the other fertilizer treatments. The R value is shown in different colors in the figure. If the P-value is < 0.05, it is marked with an asterisk. The legend on the right is the color interval of the different R-values. Cluster trees of the species and environmental factors are presented on the left and upper sides. *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; ***P ≤ 0.001.