Table_3_Elevated Atmospheric CO2 and Nitrogen Fertilization Affect the Abundance and Community Structure of Rice Root-Associated Nitrogen-Fixing Bacteria.docx

Elevated atmospheric CO2 (eCO2) results in plant growth and N limitation, yet how root-associated nitrogen-fixing bacterial communities respond to increasing atmospheric CO2 and nitrogen fertilization (eN) during the growth stages of rice is unclear. Using the nifH gene as a molecular marker, we studied the combined effect of eCO2 and eN on the diazotrophic community and abundance at two growth stages in rice (tillering, TI and heading, HI). Quantitative polymerase chain reaction (qPCR) showed that eN had no obvious effect on nifH abundance in rice roots under either ambient CO2 (aCO2) or eCO2 treatment at the TI stage; in contrast, at the HI, nifH copy numbers were increased under eCO2 and decreased under aCO2. For rhizosphere soils, eN significantly reduced the abundance of nifH under both aCO2 and eCO2 treatment at the HI stage. Elevated CO2 significantly increased the nifH abundance in rice roots and rhizosphere soils with nitrogen fertilization, but had no obvious effect without N addition at the HI stage. There was a significant interaction [CO2 × N fertilization] effect on nifH abundance in root zone at the HI stage. In addition, the nifH copy numbers in rice roots were significantly higher at the HI stage than at the TI stage. Sequencing analysis indicated that the root-associated diazotrophic community structure tended to cluster according to the nitrogen fertilization treatment and that Rhizobiales were the dominant diazotrophs in all root samples at the HI stage. Additionally, nitrogen fertilization significantly increased the relative abundance of Methylosinus (Methylocystaceae) under eCO2 treatment, but significantly decreased the relative abundance of Rhizobium (Rhizobiaceae) under aCO2 treatment. Overall, the combined effect of eN and eCO2 stimulates root-associated diazotrophic methane-oxidizing bacteria while inhibits heterotrophic diazotrophs.

大气CO₂浓度升高（elevated atmospheric CO₂, eCO₂）会促进植物生长并引发氮素限制，但目前尚不明确水稻生长周期内，根系相关固氮细菌群落如何响应大气CO₂浓度升高与氮肥施用（elevated nitrogen fertilization, eN）。本研究以固氮酶基因nifH（nifH gene）作为分子标记，探究了eCO₂与eN配施对水稻两个生长时期（分蘖期TI、抽穗期HI）固氮微生物群落组成与丰度的影响。定量聚合酶链式反应（quantitative polymerase chain reaction, qPCR）结果显示：分蘖期时，无论背景大气CO₂（ambient CO₂, aCO₂）还是eCO₂处理下，氮肥施用均未对水稻根内nifH基因丰度产生显著影响；与之相反，抽穗期时，eCO₂处理下nifH基因拷贝数显著升高，而aCO₂处理下则显著降低。对于根际土壤，抽穗期时aCO₂与eCO₂处理下，氮肥施用均显著降低了nifH基因丰度。在抽穗期，eCO₂仅在配施氮肥的条件下，可显著提升水稻根内与根际土壤的nifH基因丰度，不施氮肥时则无显著影响。抽穗期根际区域的nifH基因丰度存在CO₂与氮肥施用的显著交互效应。此外，水稻根内的nifH基因拷贝数在抽穗期显著高于分蘖期。测序分析结果表明，根系相关固氮微生物群落结构会依据氮肥施用处理发生显著聚类，且抽穗期所有根样中的优势固氮菌均为根瘤菌目（Rhizobiales）。另外，eCO₂处理下氮肥施用显著提升了甲基孢囊菌属（Methylosinus，隶属于甲基孢囊菌科Methylocystaceae）的相对丰度；而aCO₂处理下氮肥施用则显著降低了根瘤菌属（Rhizobium，隶属于根瘤菌科Rhizobiaceae）的相对丰度。总体而言，eN与eCO₂的协同作用会促进根系相关固氮型甲烷氧化菌的丰度，同时抑制异养型固氮微生物的生长。