渭北旱塬长期施肥试验中氨氧化细菌的多样性及群落结构分析
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摘要
氨氧化细菌(ammonia-oxidizing bacteria,AOB)是一类能够将氨氧化为亚硝酸盐的细菌,广泛分布于几乎所有土壤、淡水和海洋环境中。氨氧化细菌催化的亚硝化过程为硝化作用的限速步骤,在自然界氮素地球化学循环过程中起着重要的作用,因此在微生物生态学中,已经建议将其作为一种模式生物。近年来人们对黄土高原地区农业土壤的研究主要集中在长期施肥对土壤中含水量、土壤肥力、农作物产量等的影响。由于氨氧化细菌的数量和种类对土壤中氮素的转化有着重要的作用,本实验对陕西省长武县长期施肥定位试验的土壤样品进行研究,探讨长期施肥对土壤中氨氧化细菌的多样性和群落结构组成的影响,以揭示长期施肥作用下黄土高原地区农业土壤中氨氧化细菌的多样性及群落结构特点,为制定合理的施肥制度提供理论基础。
本试验样品采集于中国科学院长武农业生态站长期定位施肥基地。试验采用完全随机区组设计方案,设置5个处理:LD(裸地);CK(种植不施肥);N(单施氮肥);P(单施磷肥);NP(氮磷配施)。各处理每种肥料的施入量为:N肥(尿素)120 kg/hm~2,P肥(过磷酸钙)60 kg/hm2。
采用直接法提取土样中微生物总DNA,使用氨氧化细菌的功能基因氨单加氧酶基因(amoA)所设计的引物扩增基因片段,建立氨氧化细菌的克隆文库,用Rsa I和Hha I限制性内切酶对氨氧化细菌的克隆文库进行PCR-RFLP分析。
通过对5种长期施肥处理氨氧化细菌amoA基因克隆文库的构建及群落多样性的系统发育分析表明:
(1)从LD、CK、N、P和NP等5种施肥处理的土壤样品中分别得到38/154、48/150、65/152、45/152、46/152个RFLP酶切类型。库容值分别为86.36%、80.67%、73.68%、84.87%和81.57%。各处理均出现了明显的优势菌群。
(2)α多样性指数表明不同施肥处理的多样性存在差异,N处理的土壤中氨氧化细菌的Shannon-Wiener(H′)和Margalef(d_(Ma))指数均最高,其次是CK、NP、P处理,而LD处理中最低,表明长期单施氮肥后增加了土壤中氨氧化细菌的多样性和丰富度;长期种植作物后也同样会增加土壤中氨氧化细菌的多样性和丰富度,但单施磷肥和氮磷共施后土壤中氨氧化细菌的多样性和丰富度都有所降低,长期施肥均改变了氨氧化细菌的多样性和丰富度。
(3)基于amoA基因建立的系统进化树显示,所有来自于各处理条件下土壤中氨氧化细菌的优势种群都是属于Nitrosospira和Nitrosospira-like,与大部分与Nitrosospiracluster 3聚为一组,但优势菌种在克隆文库中所占的比例不同,表明不同的施肥处理下土壤中氨氧化细菌的群落结构发生了改变。
Ammonia-oxidizing bacteria (ammonia-oxidizing bacteria, AOB) are a type of bacteria which can oxidize ammonia to nitrite, and they are widely distributed in almost all soil, freshwater and marine environments. The process that ammonia-oxidizing bacteria catalyzed nitrosation is rate-limiting step of nitrification, and it plays an important role in natural geochemical cycling of nitrogen. Therefore in microbial ecology, AOB has been proposed as a model organism. In recent years, the agricultural soil study of the Loess Plateau is mainly focused on long-term fertilization effects on soil moisture, soil fertility, and crop yield. AOB’S number and types play an important role in nitrogen transformation. The study materials are on long-term fertilized soil samples from Changwu County, Shaanxi. The purpose is to explore the effects of long-term fertilization on the diversity of ammonia oxidizing bacteria and community structure composition and in order to reveal the characteristic of AOB’diversity and community structure in long-term fertilized agricultural soil in Loess Plateau, finally, provide a theoretical basis for reasonable fertilization.
The test samples are collected from Changwu Agricultural Ecological Station, Chinese Academy of long-term fertilization base. The experiment using a completely random design method with 5 treatments: LD (bare land); CK (cultivation without fertilization); P(superphosphate); N(urea); NP(superphosphate+urea). Fertilizer amount in each treatment is: N fertilizer (urea) 120 kg/hm2, P fertilizer (superphosphate) 60 kg/hm~2.
The total DNA of soil microorganisms was directly extracted from soil samples. Primers which were designed according to the functional gene, ammonia monooxygenase gene (amoA) were used to establish five clone libraries of ammonia-oxidizing bacteria. The clone libraries was analyzed by PCR-RFLP with cleavaging of Rsa I and Hha I, two four-base restriction enzymes, respectively.
Through construction of amoA gene library ammonia-oxidizing bacteria from five treatment soil samples and phylogenetic analysis of community diversity, it showed that:
(1) Different RFLP types by enzyme digestion were obtained from five kinds of fertilized soils. They are 38/154(LD), 48/150(CK), 65/152(N), 45/152(P) and 46/152(NP), respectively. Storage capacity values were 86.36%, 80.67%, 73.68%, 84.87% and 81.57% , respectively. In all treatments, there is a clear advantage flora.
(2)αdiversity index showed that the diversity of different treated soils was various. In N treatment soil, Shannon-Wiener (H’) and Margalef (d_(Ma)) index of ammonia-oxidizing bacteria was the highest, then followed by CK, NP, P treatment, and the lowest was the LD treatment. These indicated that after long-term N fertilizer’s treatment, the diversity and richness of ammonia oxidizing bacteria had been increased. At the same time, long-term cultivation of crops can also increase ammonia oxidizing bacterial diversity and richness. However, in single P and N and N, P mixed treated soil, the diversity and richness were reduced. These indicated that long-term fertilization changed the diversity and richness of ammonia-oxidizing bacteria.
(3) Phylogenetic tree based on amoA gene showed that the dominant ammonia oxidizing bacteria in all treated solids, belongs to Nitrosospira and Nitrosospira-like, and most of dominant bacteria are grouped with Nitrosospira cluster 3. But the dominant strain’s proportion in clone library was different, indicating that in the soils under different fertilization treatments, the structure of ammonia oxidizing bacterial community had been changed.
本试验样品采集于中国科学院长武农业生态站长期定位施肥基地。试验采用完全随机区组设计方案,设置5个处理:LD(裸地);CK(种植不施肥);N(单施氮肥);P(单施磷肥);NP(氮磷配施)。各处理每种肥料的施入量为:N肥(尿素)120 kg/hm~2,P肥(过磷酸钙)60 kg/hm2。
采用直接法提取土样中微生物总DNA,使用氨氧化细菌的功能基因氨单加氧酶基因(amoA)所设计的引物扩增基因片段,建立氨氧化细菌的克隆文库,用Rsa I和Hha I限制性内切酶对氨氧化细菌的克隆文库进行PCR-RFLP分析。
通过对5种长期施肥处理氨氧化细菌amoA基因克隆文库的构建及群落多样性的系统发育分析表明:
(1)从LD、CK、N、P和NP等5种施肥处理的土壤样品中分别得到38/154、48/150、65/152、45/152、46/152个RFLP酶切类型。库容值分别为86.36%、80.67%、73.68%、84.87%和81.57%。各处理均出现了明显的优势菌群。
(2)α多样性指数表明不同施肥处理的多样性存在差异,N处理的土壤中氨氧化细菌的Shannon-Wiener(H′)和Margalef(d_(Ma))指数均最高,其次是CK、NP、P处理,而LD处理中最低,表明长期单施氮肥后增加了土壤中氨氧化细菌的多样性和丰富度;长期种植作物后也同样会增加土壤中氨氧化细菌的多样性和丰富度,但单施磷肥和氮磷共施后土壤中氨氧化细菌的多样性和丰富度都有所降低,长期施肥均改变了氨氧化细菌的多样性和丰富度。
(3)基于amoA基因建立的系统进化树显示,所有来自于各处理条件下土壤中氨氧化细菌的优势种群都是属于Nitrosospira和Nitrosospira-like,与大部分与Nitrosospiracluster 3聚为一组,但优势菌种在克隆文库中所占的比例不同,表明不同的施肥处理下土壤中氨氧化细菌的群落结构发生了改变。
Ammonia-oxidizing bacteria (ammonia-oxidizing bacteria, AOB) are a type of bacteria which can oxidize ammonia to nitrite, and they are widely distributed in almost all soil, freshwater and marine environments. The process that ammonia-oxidizing bacteria catalyzed nitrosation is rate-limiting step of nitrification, and it plays an important role in natural geochemical cycling of nitrogen. Therefore in microbial ecology, AOB has been proposed as a model organism. In recent years, the agricultural soil study of the Loess Plateau is mainly focused on long-term fertilization effects on soil moisture, soil fertility, and crop yield. AOB’S number and types play an important role in nitrogen transformation. The study materials are on long-term fertilized soil samples from Changwu County, Shaanxi. The purpose is to explore the effects of long-term fertilization on the diversity of ammonia oxidizing bacteria and community structure composition and in order to reveal the characteristic of AOB’diversity and community structure in long-term fertilized agricultural soil in Loess Plateau, finally, provide a theoretical basis for reasonable fertilization.
The test samples are collected from Changwu Agricultural Ecological Station, Chinese Academy of long-term fertilization base. The experiment using a completely random design method with 5 treatments: LD (bare land); CK (cultivation without fertilization); P(superphosphate); N(urea); NP(superphosphate+urea). Fertilizer amount in each treatment is: N fertilizer (urea) 120 kg/hm2, P fertilizer (superphosphate) 60 kg/hm~2.
The total DNA of soil microorganisms was directly extracted from soil samples. Primers which were designed according to the functional gene, ammonia monooxygenase gene (amoA) were used to establish five clone libraries of ammonia-oxidizing bacteria. The clone libraries was analyzed by PCR-RFLP with cleavaging of Rsa I and Hha I, two four-base restriction enzymes, respectively.
Through construction of amoA gene library ammonia-oxidizing bacteria from five treatment soil samples and phylogenetic analysis of community diversity, it showed that:
(1) Different RFLP types by enzyme digestion were obtained from five kinds of fertilized soils. They are 38/154(LD), 48/150(CK), 65/152(N), 45/152(P) and 46/152(NP), respectively. Storage capacity values were 86.36%, 80.67%, 73.68%, 84.87% and 81.57% , respectively. In all treatments, there is a clear advantage flora.
(2)αdiversity index showed that the diversity of different treated soils was various. In N treatment soil, Shannon-Wiener (H’) and Margalef (d_(Ma)) index of ammonia-oxidizing bacteria was the highest, then followed by CK, NP, P treatment, and the lowest was the LD treatment. These indicated that after long-term N fertilizer’s treatment, the diversity and richness of ammonia oxidizing bacteria had been increased. At the same time, long-term cultivation of crops can also increase ammonia oxidizing bacterial diversity and richness. However, in single P and N and N, P mixed treated soil, the diversity and richness were reduced. These indicated that long-term fertilization changed the diversity and richness of ammonia-oxidizing bacteria.
(3) Phylogenetic tree based on amoA gene showed that the dominant ammonia oxidizing bacteria in all treated solids, belongs to Nitrosospira and Nitrosospira-like, and most of dominant bacteria are grouped with Nitrosospira cluster 3. But the dominant strain’s proportion in clone library was different, indicating that in the soils under different fertilization treatments, the structure of ammonia oxidizing bacterial community had been changed.
引文
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