State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, 210008, Jiangsu Province, China.
College of agricultural science and engineering, Hohai University, Nanjing, 210098, Jiangsu Province, China.
Microb Ecol. 2020 Nov;80(4):859-871. doi: 10.1007/s00248-020-01570-1. Epub 2020 Aug 15.
Upland soil clusters alpha and gamma (USCα and USCγ) are considered a major biological sink of atmospheric methane and are often detected in forest and grassland soils. These clusters are phylogenetically classified using the particulate methane monooxygenase gene pmoA because of the difficulty of cultivation. Recent studies have established a direct link of pmoA genes to 16S rRNA genes based on their isolated strain or draft genomes. However, whether the results of pmoA-based assays could be largely represented by 16S rRNA gene sequencing in upland soils remains unclear. In this study, we collected 20 forest soils across China and compared methane-oxidizing bacterial (MOB) communities by high-throughput sequencing of 16S rRNA and pmoA genes using different primer sets. The results showed that 16S rRNA gene sequencing and the semi-nested polymerase chain reaction (PCR) of the pmoA gene (A189/A682r nested with a mixture of mb661 and A650) consistently revealed the dominance of USCα (accounting for more than 50% of the total MOB) in 12 forest soils. A189f/A682r successfully amplified pmoA genes (mainly RA14 of USCα) in only three forest soils. A189f/mb661 could amplify USCα (mainly JR1) in several forest soils but showed a strong preferential amplification of Methylocystis and many other type I MOB groups. A189f/A650 almost exclusively amplified USCα (mainly JR1) and largely discriminated against Methylocystis and most of the other MOB groups. The semi-nested PCR approach weakened the bias of A189f/mb661 and A189f/A650 for JR1 and balanced the coverage of all USCα members. The canonical correspondence analysis indicated that soil NH-N and pH were the main environmental factors affecting the MOB community of Chinese forest soils. The RA14 of the USCα group prefers to live in soils with low pH, low temperature, low elevation, high precipitation, and rich in nitrogen. JR1's preferences for temperature and elevation were opposite to RA14. Our study suggests that combining the deep sequencing of 16S rRNA and pmoA genes to characterize MOB in forest soils is the best choice.
旱地土壤聚类 alpha 和 gamma（USCα 和 USCγ）被认为是大气甲烷的主要生物汇，通常在森林和草原土壤中检测到。这些聚类使用颗粒状甲烷单加氧酶基因 pmoA 进行系统发育分类，因为它们难以培养。最近的研究基于分离菌株或草案基因组，建立了 pmoA 基因与 16S rRNA 基因的直接联系。然而，旱地土壤中基于 pmoA 的测定结果是否可以很大程度上由 16S rRNA 基因测序来代表仍不清楚。在这项研究中，我们收集了中国各地的 20 个森林土壤样本，并通过使用不同引物对的 16S rRNA 和 pmoA 基因高通量测序比较了甲烷氧化菌（MOB）群落。结果表明，16S rRNA 基因测序和 pmoA 基因的半巢式聚合酶链反应（PCR）（A189/A682r 嵌套与 mb661 和 A650 的混合物）一致揭示了 12 个森林土壤中 USCα 的优势（占总 MOB 的 50%以上）。A189f/A682r 仅在三个森林土壤中成功扩增了 pmoA 基因（主要是 USCα 的 RA14）。A189f/mb661 可以在几个森林土壤中扩增 USCα（主要是 JR1），但对 Methylocystis 和许多其他 I 型 MOB 群具有强烈的优先扩增作用。A189f/A650 几乎只扩增 USCα（主要是 JR1），并大大排除了 Methylocystis 和大多数其他 MOB 群。半巢式 PCR 方法减弱了 A189f/mb661 和 A189f/A650 对 JR1 的偏倚，并平衡了所有 USCα 成员的覆盖范围。典范对应分析表明，土壤 NH-N 和 pH 是影响中国森林土壤 MOB 群落的主要环境因素。USCα 组的 RA14 更喜欢生活在 pH 值低、温度低、海拔低、降水丰富、氮丰富的土壤中。JR1 对温度和海拔的偏好与 RA14 相反。我们的研究表明，结合 16S rRNA 和 pmoA 基因的深度测序来描述森林土壤中的 MOB 是最佳选择。
