重庆紫色水稻土中“全程”和“半程”氨氧化微生物的垂直分异

Vertical abundance variations of “incomplete ammonia oxidizers” and “comammox” in purple paddy soil in Chongqing

摘要:

[目的]系统评估全程氨氧化细菌（complete ammonia oxidizing bacteria，Comammox bacteria）、半程氨氧化细菌（AOB）和古菌（AOA）在典型水稻土剖面的垂直分异规律。2015年发现的"全程"氨氧化细菌（Comammox Nitrospira）可将氨分子一步氧化为硝酸盐，实现硝化作用。而经典的"半程"氨氧化细菌（AOB）或古菌（AOA）将氨分子氧化为亚硝酸盐后，再由系统发育完全不同的硝化细菌将其氧化为硝酸盐。全程氨氧化细菌实现了一步硝化全过程，根本改变了学术界对2类微生物分步硝化的经典认知，但相关研究仍处于初步阶段。[方法]选择重庆北碚地区2017年典型水稻土并采集5、10、20和40 cm不同深度土壤（剖面采样点的上下误差不超过1 cm），提取水稻土总DNA后，利用标靶功能基因amoA，通过实时荧光定量PCR技术分析全程氨氧化细菌（Comammox）、半程氨氧化细菌（AOB）和古菌（AOA）在水稻土不同深度的数量变异规律。[结果]半程氨氧化细菌AOB和古菌AOA均随土壤深度增加呈显著下降趋势。然而，全程氨氧化细菌的两大类微生物则表现出相反的规律，Comammox Clade A的丰度随着土壤剖面的加深而显著增加（P<0.05），但Clade B并未有类似规律。Clade A在水稻土不同层次的土层中均比Clade B高出1个数量级，在5 cm和40 cm处的最低和最高值分别为3.42×10⁷、8.46×10⁷ copies/g。AOA与AOB的丰度大致相当，5 cm剖面处数量最高分别为1.23×10⁷、1.83×105 copies/g，但其平均丰度远低于全程氨氧化细菌，Comammox与AOA、AOB amoA功能基因拷贝数之比为10-2000。[结论]全程氨氧化细菌（Comammox bacteria）广泛分布于水稻土不同土层中，且数量远高于"半程"氨氧化细菌和古菌，意味着Comammox可能在水稻土硝化作用中起重要作用。

Abstract:

[Objective] Nitrification has been thought to be as a two-step process, where ammonia-oxidizing bacteria and archaea (AOB and AOA) first oxidize ammonia to nitrite, which nitrite-oxidizing bacteria (NOB) subsequently transfer to nitrate. Recently, the ability to oxidize ammonia has also been discovered in members of the genus Nitrospira, which were formerly supposed to only be capable of nitrite oxidation. The discovery of these bacteria that oxidize ammonia to nitrate (complete ammonia oxidizing bacteria, Comammox bacteria), refuted the dogma that the oxidation of ammonia and nitrite requires two distinct groups of microorganisms. The discovery of one-step nitrification and Comammox Nitrospira triggered many important scientific issues of the global nitrogen cycle, but relevant studies were still in the preliminary stage.[Methods] Using real-time quantitative PCR (Q-PCR), we characterized the distribution patterns of functional amoA gene abundances of Comammox (Clade A and Clade B), ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) at 5, 10, 20, and 40 cm soil depths (up-down error within 1 cm) of flooded paddy field (FPF) in Beibei, Chongqing in 2017.[Results] Quantitative PCR (Q-PCR) revealed that soil depth had a significant effect on different nitrifiers. Comammox Nitrospira were ecologically widespread and numerically abundant in all depths of the standard profile. The Comammox Clade A amoA gene copies were higher at deeper layers, while Comammox Clade B didn't show the same trends and its abundances varied between 1.85×10⁶ copies/g and 3.26×10⁶ copies/g in different depths of the standard profile. However, the abundance of Comammox Clade A was about to an order of magnitude more abundant than Comammox Clade B in paddy soils. On the contrary, the amoA gene abundances of AOA and AOB significantly decreased with increasing depth (P<0.05), both AOA and AOB amoA gene abundances were highest in the top layer (5 cm, 1.23×10⁷ copies/g and 1.83×10⁵ copies/g, respectively). The ratio of abundances of Comammox amoA genes to those of AOA and AOB increased significantly with the increase of soil depth, ranged from 10 to 2000.[Conclusion] The complete ammonia oxidizing bacteria (Comammox bacteria) are widely distributed in all soil depths of paddy soil and their abundances were significantly higher than "incomplete ammonia oxidizers (AOA, AOB)", which implicated that Comammox cannot be neglected in rice soil ecosystem.