Stres Blaz, Mahne Ivan, Avgustin Gorazd, Tiedje James M
Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824-1325, USA.
Appl Environ Microbiol. 2004 Jan;70(1):301-9. doi: 10.1128/AEM.70.1.301-309.2004.
The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mean) = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.
探究了标准农业管理对天然土壤和耕作土壤中反硝化原核生物一氧化二氮还原酶（nosZ）片段遗传异质性的影响。从两种土壤管理条件下分别采集了36个土壤核心样本并进行混合。对三份重复样本中的nosZ基因片段进行扩增，PCR产物经克隆后通过限制性片段长度多态性（RFLP）进行筛选。总nosZ RFLP图谱的相似度随土壤样本量增加而提高，直至3克的三份重复样本产生的RFLP图谱在视觉上对每种处理而言都相同。在天然土壤和耕作土壤之间观察到总nosZ图谱存在很大差异。对至少出现两次的主要克隆组的片段以及四个具有独特RFLP模式的随机选择克隆进行测序，以验证nosZ的身份。来自耕作农田的nosZ克隆的序列多样性更高，在从两种土壤中鉴定出的182种不同的nosZ RFLP模式中，两种土壤的克隆文库中仅发现了8种模式。一组占所有克隆32%的克隆在天然土壤的基因文库中占主导地位，而在耕作土壤的基因文库中观察到许多小的克隆组。nosZ RFLP数据的Chao1非参数丰富度估计值的95%置信区间不重叠，表明两种土壤中的物种丰富度水平存在显著差异，耕作土壤具有更高的多样性。对推导的氨基酸序列进行系统发育分析，将大多数nosZ克隆归入一个交错的密歇根土壤簇，其培养成员为α-变形菌纲。来自耕作土壤的只有四个nosZ序列和来自天然土壤的一个序列与γ-变形菌纲中发现的序列相关。来自天然农田的序列形成了一个独特的、密切相关的簇（平均距离D = 0.16），包含91.6%的天然克隆。来自耕作农田的克隆彼此之间的亲缘关系更远（平均距离D = 0.26），65%的克隆在来自天然土壤的簇之外，进一步表明了两个群落之间的差异。总体而言，土壤的使用与nosZ序列的丰富度、多样性以及系统发育位置之间似乎存在一种关系，表明土壤的农业利用导致了向更多样化的反硝化群落的转变。
