关键词: 固氮微生物, 秸秆还田, 固氮速率, ¹⁵N₂定量, nifH

Abstract:

【Objective】The purpose of this study was to analyze the effects of different straw additions on soil N₂-fixation rate and diazotroph community structure, which was crucial for the management of crop residue and mineral fertilizer application in China.【Method】Using indoor incubation experiment, in addition to the control (C0: 0), a total of 5 straw addition gradients (C1: 0.2 mg·g^-1; C2: 1.0 mg·g^-1; C3: 2.0 mg·g^-1; C4: 4.0 mg·g^-1; C5: 10.0 mg·g^-1), using the ¹⁵N₂ labeling method, and soil samples were collected after 28 days of incubation in dark conditions. As a direct measure of N₂-fixation,¹⁵N₂ gas labeling method could quantify the rate of biological N₂-fixation, and destined for characterization of nifH gene marker and diazotroph community by using the Illumina PE250 sequencing and PCR techniques. 【Result】With the increase of straw addition, the content of NO₃^--N in soil decreased significantly, while the content of NH₄⁺-N did not change significantly. The pH of soil decreased and the rate of biological N₂-fixation increased significantly. C3, C4 and C5 treatments could reach 2.71-3.05 μg N·g^-1DW during the incubation period (28 d), which was about 87-96 kg N·hm^-2·a^-1, compared with C0, the rate of biological N₂-fixation increased by 38.1%-52.4%. The copy number of nifH gene ranged from 5.48×10⁷ to 9.20×10⁷ copies/(g soil) under all treatments. Compared with C0, the copy number of nifH gene was significantly increased under C4 and C5 treatments, and the number and activity of diazotroph were significantly increased (P<0.05). However, when the amount of straw added reached 4.0 mg·g ^-1, increasing the amount of straw had no significant effect on the copy number of nifH gene of soil diazotroph. With the increase of straw addition, Shannon-Weill index of C4 and C5 levels was significantly lower than that of the other four treatments (P<0.05), and the diversity of the other four treatments had no significant difference. The result of PCoA showed that diazotroph community structure was clustered into different groups depending upon the difference of straw addition. Diazotroph were divided into Proteobacteria, Cyanobacteria, Firmicutes, Actinobacteria and Spirochaetes at the phylum level. As the amount of straw added increases, the relative abundance of Cyanobacteria tends to increase first and then decrease. At the genus level, there were significant differences in the number of carbon sources among different species. Bradyrhizobium was the most abundant genus. With the increase of straw addition, the relative abundance of C5 was significantly different from that of C0, C1, C2 and C3 (P<0.05), increasing by 12.07%-14.13%. The relative abundance of Scytonema hofmanni, which was positively correlated with the rate of biological N₂-fixation, gradually increases with the increase of straw addition, but decreased when straw addition reaches C5 level (P<0.05). Meanwhile, PLS-PM analysis, RDA and correlation analysis revealed that the rate of biological N₂-fixation and soil diazotroph community were greatly affected by straw addition and soil NO₃^--N concentration. 【Conclusion】The rate of soil biological N₂-fixation increases with the amount of straw added. The effect of straw on N₂-fixation was mainly due to the reduction of NO₃^--N content in the soil by straw addition, which in turn promoted the diazotroph such as Bradyrhizobium, Scytonema hofmanni and Azospirillum grow. According to the calculation results of this experiment, compared with no straw, after the straw was added (4.0 mg·g ^-1), the rate of biological N₂-fixation could be increased by 26 kg N·hm^-2·a^-1, which significantly reduced the fertilizer demand.

Key words: soil diazotroph, straw returning, the rate of biological N₂-fixation, ¹⁵N₂ labeling method, nifH