Effect of endophytic diazotroph Enterobacter roggenkampii ED5 on nitrogen-metabolism-related microecology in the sugarcane rhizosphere at different nitrogen levels

Abstract

Sugarcane is an important sugar and energy crop worldwide, requiring a large amount of nitrogen (N). However, excessive application of synthetic N fertilizer causes environmental pollution in farmland. Endophytic nitrogen-fixing bacteria (ENFB) provide N nutrition for plants through biological N fixation, thus reducing the need for chemical fertilizers. The present study investigated the effect of the N-fixing endophytic strain Enterobacter roggenkampii ED5 on phytohormone indole-3-acetic acid (IAA), N-metabolism enzyme activities, microbial community compositions, and N cycle genes in sugarcane rhizosphere soil at different N levels. Three levels of ¹⁵N-urea, such as low N (0 kg/ha), medium N (150 kg/ha), and high N (300 kg/ha), were applied. The results showed that, after inoculating strain ED5, the IAA content in sugarcane leaves was significantly increased by 68.82% under low N condition at the seedling stage (60 days). The nitrate reductase (NR) activity showed a downward trend. However, the glutamine synthase (GS) and NADH-glutamate dehydrogenase (NADH-GDH) activities were significantly enhanced compared to the control under the high N condition, and the GS and NR genes had the highest expression at 180 and 120 days, respectively, at the low N level. The total N content in the roots, stems, and leaves of sugarcane was higher than the control. The ¹⁵N atom % excess of sugarcane decreased significantly under medium N condition, indicating that the medium N level was conducive to N fixation in strain ED5. Metagenome analysis of sugarcane rhizosphere soil exhibited that the abundance of N-metabolizing microbial richness was increased under low and high N conditions after inoculation of strain ED5 at the genus level, while it was increased at the phylum level only under the low N condition. The LefSe (LDA > 2, p < 0.05) found that the N-metabolism-related differential microorganisms under the high N condition were higher than those under medium and low N conditions. It was also shown that the abundance of nifDHK genes was significantly increased after inoculation of ED5 at the medium N level, and other N cycle genes had high abundance at the high N level after inoculation of strain ED5. The results of this study provided a scientific reference for N fertilization in actual sugarcane production.

Keywords: Enterobacter roggenkampii ED5; metagenomics; nitrogen fixation; nitrogen levels; rhizosphere; sugarcane growth.

Figure 1

Heatmap analysis of the trend of total nitrogen content changes in sugarcane GT11 inoculated with E. roggenkampii ED5. (A) Root (N%); (B) Stem (N%); (C) Leaf (N%). The color intensity in each panel of the heatmap showed the total nitrogen content of sugarcane GT11.

Figure 2

Effect of E. roggenkampii ED5 on the IAA contents and activities of N-metabolism-related enzymes in leaves of sugarcane GT11 at different nitrogen application levels. (A) IAA; (B) NR; (C) GS; (D) NADH-GDH. The same letter indicates no significant difference was detected at Duncan's multiple range test, P ≤ 0.05 (n = 3).

Figure 3

Expression of NR and GS genes in leaves of sugarcane GT11 at different nitrogen application levels. (A) GS; (B) NR. The same letter indicates no significant difference was detected at Duncan's multiple range test, P ≤ 0.05 (n = 3).

Figure 4

N-metabolism-related microbial community structures in sugarcane variety GT11 rhizosphere soil at different nitrogen application levels. (A, B) On the phylum level; (C, D), on the genus level.

Figure 5

The LEfSe bar chart of N-metabolism-related differences in taxon abundance in the rhizosphere soil of sugarcane GT11 at different nitrogen levels.

Figure 6

Heatmap analysis showing functions of N-metabolism-related microorganisms in the rhizosphere soil of sugarcane GT11 at different nitrogen application levels. The color intensity in each panel of the heatmap showed the functional abundance of KO.

Figure 7

The abundance of nitrogen fixation functional genes in the rhizosphere of sugarcane GT11. (A) Nitrogenase genes; (B) nitrification process genes; (C) denitrification process genes; (D) nitrate assimilation and reduction to ammonia-related genes. The same letter indicates no significant difference was detected at Duncan's multiple range test, P ≤ 0.05 (n = 3).