阔叶林改种毛竹(Phyllostachys pubescens)后土壤固氮细菌 nifH 基因多样性的变化

【目的】毛竹是喜氮植物,土壤氮素水平对毛竹生长至关重要。生物固氮是土壤氮素的重要来源,因此,探索阔叶林改种毛竹后土壤固氮细菌和土壤氮素的变化具有重要意义。【方法】选择立地条件相近的毛竹林(100多年前由阔叶林改种而来)和阔叶林,每种林地在东北坡向位置随机选择4个10 m×10 m标准样地,每个标准样地选取5个采样点,分层采集0—20 cm(表层)和20—40 cm(次表层)土壤样品,分析了土壤pH、有机碳、碱解氮、有效磷、速效钾和含水量等常规理化性质; 采用引物对AQER和PolF,以土壤总DNA为模板扩增了固氮细菌功能基因(nifH )片段,应用变性梯度凝胶电泳(DGGE)和实时荧光定量PCR(Real-time PCR),分析了固氮细菌群落结构、多样性以及丰度(nifH 基因拷贝数)变化; 通过基因克隆测序对土壤固氮细菌进行初步鉴定。【结果】阔叶林改种毛竹后土壤pH显著(P0.05)提高; 毛竹林土壤的含水量、碱解氮以及表层土壤的速效钾显著高于(P0.05)同层的阔叶林土壤,而有效磷则显著(P0.05)低于同层的阔叶林土壤。总体来说,阔叶林改种毛竹后土壤养分含量明显提高; 阔叶林土壤固氮细菌DGGE条带数以及多样性指数(Shannon-Wiener index)都高于毛竹林; 基于DGGE条带信息的聚类分析和主成分分析(PCA)结果表明,阔叶林和毛竹林区分为2个类群,而同一林分的不同土层之间差异较小; 实时荧光定量PCR结果显示,毛竹林土壤的固氮细菌 nifH 基因丰度显著(P0.05)高于阔叶林土壤; 通过克隆测序,14个阳性克隆分别属于2个不同的菌属,其中13个均为Bradyrhizobium,1个为Azohydromonas lata,条带序列与已知序列的相似度为93%~98%。【结论】阔叶林改种毛竹后土壤固氮细菌的种类减少,而功能基因丰度却明显增加; 土壤氮素水平明显提高,这可能是土壤固氮能力增强的结果。

Variation of nifH gene diversity of soil nitrogen-fixing bacteria in Moso bamboo (Phyllostachys pubescens) plantation converted from broadleaf forest

【Objectives】 Nitrogen plays an important role in promoting the growth of Moso bamboo, the major source of nitrogen in forest soil is derived from biological nitrogen fixation. However, little is known about the impact of conversion of broadleaf forest to bamboo (Phyllostachys pubescens) plantation on changes in soil nitrogen contents and soil nitrogen-fixing bacterial community structure.【Methods】 Two plots of broadleaf forest and Moso bamboo stands with the same site condition were selected. And 4 standard areas (10 m×10 m) were chosen randomly on the northeast slope in each plot. Soil samples were collected from the topsoil (0-20 cm) and subsoil (20-40 cm) layers separately. Soil pH, organic matter, available N, available P and readily available K were analyzed. The structure and abundance of nitrogen-fixing bacterial community were measured withprimers AQER and PolF via denaturing gradient gel electrophoresis(DGGE) and qPCR based on nifH gene. Preliminary identifying of soil nitrogen-fixing bacteria was sequenced by cloning. 【Results】The results showed that compared to the broadleaf forest, Moso bamboo stands had significant higher pH, moisture, contents of available N in both topsoil and subsoil and readily available K in topsoil, while the soil available P was much lower. The DGGE profiles indicated that Moso bamboo stands had lower numbers of band and Shannon-Wiener index (H). Both cluster analysis and principal components analysis (PCA) of the DGGE profiles showed the separation of the two soils clearly, and there was little difference between topsoil and subsoil for the same treatment. The real-time PCR analysis revealed that copy number of nifH gene in Moso bamboo was higher than that in broadleaf forest. Sequencing of 14 DGGE bands isolated revealed that 13 of them were affiliated with Bradyrhizobium and 1 with Azohydromonas, with the similarities ranging from 93% to 98% in known species of Gene Bank.【Conclusions】 This results showed that the conversion of broadleaf forest to bamboo plantation decreased the diversity of nitrogen-fixing bacterial community, but increased its abundance and nitrogen contents, indicating an enhanced nitrogen-fixing ability of soil.