应用454测序技术分析种植制度对黑垆土微生物多样性的影响

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蔡艳, 郝明德, 张丽琼, 臧逸飞, 何晓雁. 应用454测序技术分析种植制度对黑垆土微生物多样性的影响. 41(2): 339-346
CAI Yan, HAO Ming-De, ZHANG Li-Qiong, ZANG Yi-Fei, HE Xiao-Yan. Effect of Cropping Systems on Microbial Diversity in Black Loessial Soil Tested by 454 Sequencing Technology. Acta Agronomica Sinica, 41(2): 339-346 复制到剪切板

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作物学报编辑部

应用454测序技术分析种植制度对黑垆土微生物多样性的影响

蔡艳^1,², 郝明德^1,^3,^*, 张丽琼¹, 臧逸飞¹, 何晓雁¹

¹西北农林科技大学资源环境学院, 陕西杨凌712100

²四川农业大学资源环境学院, 四川成都611130

³西北农林科技大学水土保持研究所, 陕西杨凌712100

^* 通讯作者(Corresponding author): 郝明德, E-mail: mdhao@ms.iswc.ac.cn, Tel: 029-87012322

第一作者联系方式: E-mail: caiyya@126.com, Tel: 028-86291371

收稿日期:2014-12-19

基金:本研究由国家科技支撑计划重大项目(2011BAD31B01)和宁夏农业综合开发科技推广项目(NTKJ-2013-03-1)资助;

摘要

研究典型种植制度对旱地黑垆土微生物多样性的影响, 对优化旱地作物种植制度、发挥土壤潜在肥力、实现土壤资源可持续利用有着重要的意义。通过27年长期定位试验, 采用454测序技术分析了黄土高原旱作地区不同种植制度下黑垆土细菌、真菌多样性的变化情况。结果表明, 不施肥低营养胁迫下, 细菌多样性表现为粮豆轮作>小麦连作>裸地>苜蓿连作, 真菌多样性表现为小麦连作≈苜蓿连作>裸地>粮豆轮作。施氮、磷肥条件下, 粮草长周期轮作(苜蓿→苜蓿→苜蓿→苜蓿→马铃薯→小麦→小麦→小麦+苜蓿)中土壤微生物多样性大致表现出先降低后增加的趋势, 第4年苜蓿或苜蓿茬后第1年小麦微生物Chao指数和Shannon指数最低, 苜蓿茬后第2年小麦微生物多样性最高, 细菌Chao指数和真菌Shannon指数比连作小麦高22.0%和79.2%; 粮草短周期轮作(红豆草→小麦→小麦+红豆草)中土壤微生物多样性大致呈现增加趋势, 至红豆草茬后第2年小麦土壤微生物多样性达到最高, 真菌Chao指数和Shannon指数均分别比连作小麦高50.8%和51.0%。黄土高原旱地区黑垆土采取粮食作物与豆科作物轮作可提高土壤微生物多样性。

关键词: 454测序; 高通量测序; 旱地土壤; 微生物多样性; 种植制度

Effect of Cropping Systems on Microbial Diversity in Black Loessial Soil Tested by 454 Sequencing Technology

CAI Yan^1,², HAO Ming-De^1,^3,^*, ZHANG Li-Qiong¹, ZANG Yi-Fei¹, HE Xiao-Yan¹

¹ College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, China

² College of Resources and Environment, Sichuan Agricultural University, Chengdu 611130, China

³ Institute of Soil and Water Conservation, Northwest A&F University, Yangling 712100, China

Abstract

It has important significance on optimizing dryland cropping systems, mining the soil potential fertility and achieving the sustainable use of soil resources that we study the influence of cropping systems on soil microbial diversity in typical arid areas. In the long-term experiment lasted 27 years, we analyzed the diversity changes of bacteria and fungi in black loessial soil under different cropping systems in Loess Plateau using 454 sequencing technology. The results showed that bacterial diversity showed wheat-pea rotation>continuous wheat>fallow land>continuous alfalfa, and fungal diversity showed continuous wheat≈continuous alfalfa>fallow land>wheat-pea rotation under low nutritional stress with no fertilization. In the conditions of application of nitrogen fertilizer and phosphorus fertilizer, microbial diversity generally showed an decreasing and then increasing trend in long-period rotation of wheat-alfalfa (alfalfa→alfalfa→alfalfa→alfalfa→potato→wheat→wheat→wheat, eight years as a rotation period); Chao index and Shannon index of the 4th year alfalfa or the 1st year wheat were the lowest, and those of the 2nd year wheat were the maximum, with the bacteria Chao index and fungi Shannon index of 22.0% and 79.2% higher than those of continuous wheat, respectively. Microbial diversity generally showed an increasing trend in short-period rotation of wheat-sainfoin (sainfoin→wheat→wheat, three years as a rotation period), and that of the 2nd year wheat after sainfoin was the maximum, with fungi Chao index and Shannon index of 50.8% and 51.0% higher than that of continuous wheat respectively. Wheat-forage legumes rotation could improve the microbial diversity significantly in Loess dryland areas.

Keyword: The 454 sequencing; High-throughput sequencing; Dry land soil; Microbial diversity; Cropping system

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土壤微生物是使土壤具有生命力的主要成分, 与土壤肥力和土壤健康有着密切关系, 在土壤形成与发育、物质转化和能量传递等过程中发挥着重要作用, 是评价土壤肥力的重要指标之一^[1]。土壤微生物多样性指土壤生态系统中所有的微生物种类、它们拥有的基因以及这些微生物与环境之间相互作用的多样化程度^[2]。土壤微生物多样性的研究方法很多, 但传统的筛选培养方法仅限于对环境中极少部分可培养微生物类群的研究^[3], 常规分子生物学手段, 如PCR-DGGE、RFLP和RAPD等方法亦因操作复杂、实验周期长、成本高、痕量微生物检测困难等因素无法达到深入分析的目的。高通量测序技术无需构建克隆, 且通量高, 可以大量构建基因信息库进行比较分析, 使高效、全面认识土壤微生物成为可能。该技术自2006年问世以来, 已广泛应用于植物学、动物学、医学及极端环境微生物研究^{[4, 5, 6]}。其中454测序技术因序列读长最长, 目前在土壤微生物多样性研究中应用最广泛^{[7, 8, 9, 10]}。

黑垆土是我国黄土高原地区形成的主要地带性耕作土壤之一, 广泛分布在渭北、宁南、陇东和陇中、晋西北地区, 集中分布在黄土旱塬区, 一直是黄土高原重要的农业生产和优质农产品基地, 在黄土高原地区粮食安全中具有重要地位^[11]。许多学者大量研究了黄土旱作区黑垆土上施肥和种植制度对土壤养分积累、作物产量和品质效应、水分利用效率及土壤环境效应等方面的影响^{[12, 13, 14, 15, 16, 17]}, 但结合现代分子生物学手段对其土壤微生物多样性的研究尚属空白。本文研究黄土旱地区不同施肥措施下种植制度对黑垆土微生物多样性的影响, 以期对该地区优化作物种植制度, 实现土壤资源可持续利用提供理论依据。

1 材料与方法

1.1 试验概况

试验地位于陕西省长武县十里铺村塬面旱地, 地处黄土高原中南部, 农业生产完全依赖天然降水, 属典型的旱作农业区, 一年一熟。多年平均降水量为578.5 mm, 其中7月至9月降水量占年总量的57%左右; 年平均气温9.3℃, 年日照时数2230 h。

试验开始于1984年, 供试土壤为黏化黑垆土, 母质是深厚的中壤质马兰黄土, 全剖面土质均匀疏松, 通透性好, 肥力中等。起始时土壤耕层含有机质10.50 g kg^-1、全氮0.80 g kg^-1、碱解氮37 mg kg^-1、有效磷3.0 mg kg^-1、速效钾129 mg kg^-1, pH 8.4。

试验设16个处理, 包括4个不施肥处理, 即裸地, 小麦连作, 苜蓿连作, 粮豆轮作(豌豆→ 小麦→ 小麦+糜子); 12个施氮、磷肥处理, 即小麦连作, 粮草长周期轮作(苜蓿→ 苜蓿→ 苜蓿→ 苜蓿→ 马铃薯→ 小麦→ 小麦→ 小麦+苜蓿), 粮草短周期轮作(红豆草→ 小麦→ 小麦+红豆草)。

小区长10.26 m, 宽6.5 m, 面积66.69 m²。每个处理3次重复, 随机区组排列。年施肥量为N 120 kg hm^-2, P₂O₅ 60 kg hm^-2, 所施氮肥为尿素, 磷肥为过磷酸钙, 于作物播前将肥料撒施地表后耕翻入土, 田间管理同大田。

1.2 土壤样品采集与处理

定位试验27年, 即2011年8月底采集土样(土样基本性质见表1)。使用不锈钢取土器, 在每个小区采用5点S形法采集0~15 cm土层, 3个试验小区土样组成混合样, 四分法保留约500 g。鲜土去除植物残体, 过2 mm筛后保存于-80℃, 用于微生物多样性分析。

表1 供试土样基本性质 Table 1 Basic properties of the tested soil samples

1.3 土壤微生物多样性分析

1.3.1 土壤总DNA的提取取0.50 g土样, 使用OMEGA公司生产的E.Z.N.A Soil DNA试剂盒按说明书抽提并纯化土壤微生物基因组DNA。

1.3.2 PCR扩增

(1) 细菌PCR扩增

将纯化后的基因组DNA作为PCR的模板, 采用细菌16S rRNA V1~V3区具有特征性的引物对27F和553R扩增^[18], 其序列分别为27F (5′ -AGAGT TTGATCCTGGCTCAG-3′ )和533R (5′ -TTACCGCGG CTGCTGGCAC-3′ ), 由上海美吉生物医药科技有限公司合成, 同时在前引物5′ 端添加Barcode序列区分样品。反应体系含: 5 × FastPfu缓冲液4.0 μ L、2.5 mmol L^-1 dNTPs 2 μ L、正向引物(5 μ mol L^-1) 0.4 μ L、反向引物(5 μ mol L^-1) 0.4 μ L、FastPfu聚合酶0.4 μ L、模板DNA 10 ng, 补ddH₂O至20 μ L。反应程序为95℃预变性2 min, 95℃变性30 s, 55℃退火30 s, 72℃延伸30 s, 25个循环后, 72℃ 延伸5 min, 保持10℃直到反应完成。每个样品3次重复, 将同一样品的PCR产物混合后用2%琼脂糖凝胶电泳检测, 使用AxyPrepDNA凝胶回收试剂盒(AXYGEN公司)切胶回收PCR产物, Tris-HCl洗脱, 2%琼脂糖凝胶电泳检测。

(2) 真菌PCR扩增

采用真菌18S rRNA具有特征性的引物对ITS1和ITS4扩增^{[19, 20]}, 其序列分别为ITS1 (5′ -TCCGT AGGTGAACCTGCGG-3′ ), ITS4 (5′ -TCCTCCGCTTA TTGATATGC-3′ )。反应体系含5 × FastPfu缓冲液4 μ L、2.5 mmol L^-1 dNTPs 2 μ L、正向引物(5 μ mol L^-1) 0.8 μ L、反向引物(5 μ mol L^-1) 0.8 μ L、FastPfu聚合酶0.4 μ L、模板DNA 10 ng, 补ddH₂O至20 μ L。反应程序为: 95℃预变性2 min, 95℃变性30 s, 53℃退火30 s, 72℃延伸30 s, 32个循环后, 72℃延伸5 min, 10℃直到反应完成。其他方法同细菌PCR扩增。

1.3.3 Roche Genomo Sequencer FLX+上机测序

由上海美吉生物医药科技有限公司完成。试剂采用Roche GS FLX + Sequencing Method Manual_XLR70 kit。

1.3.4 生物信息分析方法使用mothur软件完成^[21]。分析流程如下。

(1) 有效序列数据分析通过Barcode区分各样品的测序数据, 生成不含Barcode的各样品测序序列。

(2) 优化数据分析 ①保留引物的错配数在2个以内的序列; ②去除序列末端的后引物的接头序列、多碱基N、polyA/T尾巴及低质量碱基; ③去除长度短于200 bp, 模糊碱基数大于0、序列平均质量低于25的序列。共获得320 003条优化序列; 其中细菌231 937条, 每个土样7473~9514条; 真菌88 066条, 每个土样2624~9347条。

(3) OUT (Operational Taxonomic Units)聚类分析主要步骤为①提取非重复序列; ②与silva库(http://www.arb-silva.de/)(silva SSU111版)中的aligned (16S/18S, SSU)核糖体序列数据库比对, 比对方法为kmer searching^[22]; ③使用UCHIME方法检测并去除Chimeric序列; ④计算比对对齐后的序列间非校正配对距离; ⑤使用最远邻近法聚类生成OTU。共获得86 639个OTU; 其中细菌83 392个, 每个土样3225~6736个; 真菌3447个, 每个土样167~280个。

(4) 丰富度指数和多样性指数计算选取Chao指数和Shannon指数反映微生物多样性。

其中, S_obs为实际观测到的OTU数; n₁为只含有一条序列的OTU数目; n₂为只含有2条序列的OTU数目。

其中, S_obs为实际测量出的OTU数目; n_i为含有i条序列的OTU数目; N为所有的序列数。

2 结果与分析

2.1 不施肥条件下种植制度对旱地黑垆土微生物多样性的影响

长期不施肥且连续种植作物后, 作物对有效养分的选择性吸收导致土壤不同程度贫瘠化, 同时不同作物通过根系活动或残留根茬对土壤微生物产生不同影响。Chao指数是用来估计群落中物种总数的指数, Shannon指数是用来估算样品中物种多样性的指数。不施肥条件下, 旱地黑垆土微生物多样性见图1。细菌多样性表现为粮豆轮作> 小麦连作> 裸地> 苜蓿连作, 说明低营养胁迫下采取粮豆轮作最有利于提高细菌多样性, 其Chao指数比裸地高54.1%, Shannon指数也比裸地高0.37。连续种植小麦后土壤细菌Chao指数与裸地相当, 但Shannon指数提高0.12, 表明其细菌组成发生了变化。连续种植苜蓿后细菌Chao指数反而下降10.9%。

不施肥条件下, 旱地黑垆土真菌多样性表现为小麦连作≈ 苜蓿连作> 裸地> 粮豆轮作(图1), 说明低营养胁迫下连作比轮作更有利于提高真菌多样性。小麦连作真菌Chao指数比裸地提高10.3%, 小麦连作和苜蓿连作真菌Shannon指数比裸地高19.9%和35.8%, 粮豆轮作真菌Chao指数反而比裸地下降10.9%。

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	图1 不施肥下种植制度对旱地黑垆土微生物多样性的影响CK: 裸地; CW: 小麦连作; CA: 苜蓿连作; WP: 粮豆轮作。Fig. 1 Effect of cropping systems on microbial diversity in dryland soil without fertilizingCK: fallow land; CW: continuous wheat; CA: continuous alfalfa; WP: wheat-pea rotation.

2.2 施氮、磷肥下轮作对旱地黑垆土微生物多样性的影响

2.2.1 粮草长周期轮作中不同茬口土壤微生物多样性常规施肥条件下, 种植制度对土壤微生物的影响主要通过植物的选择性吸收和根系活动来实现。一般认为, 轮作比连作更有利于作物生长, 但对轮作周期中各茬口的微生物组成多样性知之甚少。从图2可以看出, 在施氮、磷肥条件下, 旱地粮草长周期轮作中不同茬口微生物多样性表现出较大差异性。在8年轮作周期中, 随着苜蓿种植时间延长, 土壤细菌物种数呈现先小幅增加后明显降低的趋势, 至第1年小麦茬(AW1)细菌Chao指数降到最低, 分别比第1年苜蓿茬(A1)和第2年苜蓿茬(A2)减少13.4%和14.4%。但至第2年小麦茬(AW2)细菌Chao指数升至最高, 达14 287, 比AW1高23.4%。各茬口细菌物种组成多样性变化趋势与物种数大致相似, A4 (第4年苜蓿茬)和AW1细菌Shannon指数最低, 仅7.79和7.85, AW2细菌Shannon指数最高, 达8.09。与连作小麦土壤(Chao指数为11 713, Shannon指数为7.87)相比, 粮草长周期轮作AW2和AW3 (第3年小麦茬)细菌多样性均有不同程度提高, 但AW1细菌多样性稍低于连作小麦。

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图2 粮草长周期轮作中不同茬口土壤微生物多样性A1: 第1年苜蓿; A2: 第2年苜蓿; A3: 第3年苜蓿; A4: 第4年苜蓿; P: 马铃薯; AW1: 第1年小麦; AW2: 第2年小麦; AW3: 第3年小麦。Fig. 2 Microbial diversity in different stubble fields in long-period rotation of wheat-alfalfaA1: alfalfa planted in the first year; A2: alfalfa planted in the second year; A3: alfalfa planted in the third year; A4: alfalfa planted in the fourth year; P: potato; AW1: wheat planted in the first year; AW2: wheat planted in the second year; AW3: wheat planted in the third year.

在施氮、磷肥条件下, 粮草长周期轮作中茬口对土壤真菌多样性的影响程度大于细菌。从图2可以看出, 种植苜蓿的4年里, 真菌多样性变化剧烈, A1真菌Chao指数最高, 至A2最低, 降幅达35.3%; 各茬苜蓿真菌Shannon指数排序为A2 (3.96) > A1 (3.51) > A3 (2.66) > A4 (1.51), A3 (第3年苜蓿)和A4真菌Shannon指数较A2分别降低32.8%和57.0%。种植苜蓿后马铃薯茬及小麦茬真菌Chao指数相差不明显, 但真菌Shannon指数大致呈现出逐年增加的趋势, AW2和AW3真菌Shannon指数分别达到最高(4.30)或次高(4.04), 表现出丰富的组成多样性。与连作小麦(Chao指数为301, Shannon指数为2.40)相比, 粮草长周期轮作各茬小麦真菌Chao指数提高9.3%~21.9%, Shannon指数提高36.7%~79.2%, 说明施氮、磷肥条件下粮草长周期轮作可明显提高小麦土壤真菌多样性。

2.2.2 粮草短周期轮作中不同茬口微生物多样性

红豆草被誉为“ 牧草皇后” , 在我国西北和华北地区畜牧业发展中颇受重视, 其与小麦短周期轮作是黄土旱塬区比较常见的栽培方式。从图3可以看出, 施氮磷肥条件下, 粮草短周期轮作中各茬口对土壤细菌多样性的影响程度大于真菌。细菌多样性指数均表现为第2年小麦茬(SW2) > 第1年小麦茬(SW1) > 红豆草茬(S), SW2细菌Chao指数比S提高14.6%。值得注意的是, 虽然种植红豆草后小麦土壤细菌多样性提高, 但仍与连作小麦大致相当, 明显低于粮草长周期轮作中AW2和AW3。真菌多样性表现为SW2> SW1≈ S。与连作小麦相比, 粮草短周期轮作中小麦真菌多样性明显提高, 其Chao指数和Shannon指数分别比连作小麦提高27.9%~50.8%和14.6%~50.8%, 且真菌Chao指数比粮草长周期轮作中各茬小麦增加4.9%~38.0%。

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	图3 粮草短周期轮作中不同茬口土壤微生物多样性S: 红豆草; SW1: 第1年小麦; SW2: 第2年小麦。Fig. 3 Microbial diversity in different stubble fields in short-period rotation of wheat-sainfoinS: sainfoin; SW1: wheat planted in the first year; SW2: wheat planted in the second year.

3 讨论

土壤微生物多样性与表层植被变化有很大关系, 因为植物凋落物或残留物中碳氮的生化组成及可利用程度和养分含量是影响土壤微生物多样性的主要因素^{[23, 24]}。Navarro-Noya等^[8]认为, 小麦-玉米等禾本科作物轮作及连作不会影响其细菌的丰富度和多样性, 但Sul等^[9]和Acosta-Martí nez等^[25]分别发现, 冬季栽培豌豆及采取棉花-小麦-玉米轮作比连作更有利于提高土壤细菌多样性, 吴凤芝等^[26]也证明黄瓜与小麦和大豆轮作可显著提高土壤微生物多样性, 说明不同种植制度使土壤温度、湿度等环境因素及土壤微生物可利用的营养发生了不同变化^{[16, 27]}, 对其中微生物多样性的影响程度也不完全相同。

不施肥低营养胁迫条件下, 土壤缺乏微生物生长所需碳源和矿质元素, 抑制其多样性。连续种植苜蓿后, 土壤有机质、全氮含量分别增加140.9%和141.2%, 碱解氮和速效钾等有效养分含量分别增加214.6%和29.2%, 与陈磊等^[13]研究结果一致, 这将有利于提高土壤微生物多样性; 但连续种植苜蓿会加速土壤水分^{[13, 16]}和磷素的耗竭^[28], 降低土壤微生物多样性。连续种植小麦后, 虽然有助于改善旱地土壤水分状况^{[13, 16]}, 但土壤有效养分含量减少, 会对部分微生物生长产生抑制作用。长期粮豆轮作不仅可丰富土壤有机质和氮素^[27], 还可利用轮作优势恢复旱地土壤水分, 故可明显提高旱地土壤细菌多样性。粮豆轮作土壤真菌多样性不及作物连作, 可能与低营养胁迫下微生物之间竞争有限的营养有很大关系^[29]。

在施氮磷化肥条件下, 粮草长周期轮作中细菌和真菌多样性均大致表现出先降低后增加的趋势, 第4年苜蓿茬及种植苜蓿后第1年小麦茬土壤微生物多样性较低, 可能仍与连续种植苜蓿后土壤干燥化加剧^{[13, 16]}, 不利于微生物生长繁殖有关。苜蓿茬后种植小麦, 土壤水分逐渐恢复^{[13, 16]}, 至第2年小麦茬土壤微生物多样性最高, 且明显高于连作小麦, 此时最有利于发挥土壤潜在肥力, 协调植物养分供应, 提高作物产量和品质^{[14, 15]}。Sul等^[9]认为, 有机碳是造成土壤微生物多样性差异的主要因素。种植红豆草更有利于土壤有机质累积^[27], 故其后细菌、真菌多样性大致呈现出增加的趋势, 至红豆草茬后第2年小麦土壤微生物多样性达到最高。值得一提的是, 苜蓿和红豆草虽均为豆科牧草, 但苜蓿对后茬小麦土壤细菌多样性的提升作用较明显, 红豆草则对真菌效果更突出, 生产上可适当考虑以多茬轮作模式提高土壤微生物多样性, 进而提高土壤养分转化效率。

樊军等^[30]采用稀释平板法测定种植制度对旱地土壤微生物菌群的影响, 发现低营养条件下土壤可培养微生物表现为小麦连作> 粮豆轮作> 裸地> 苜蓿连作, 粮草短周期轮作中红豆草> 第1年小麦> 第2年小麦, 与本文研究结果不一致。由于人类可培养的微生物数量大约仅占土壤微生物总数1%左右^[3], 我们认为本研究结果更能真实反映种植制度对旱地土壤微生物的影响状况。还应该进一步利用测序结果对微生物进行分类研究, 并结合土壤管理措施及理化性质, 充分揭示黄土旱塬区典型种植制度对土壤微生物类群尤其是功能微生物类群的影响。

4 结论

不施肥低营养胁迫下, 粮豆轮作有利于提高细菌多样性, 苜蓿连作和小麦连作则有利于提高真菌多样性。施肥条件下, 粮草长周期轮作有利于提高小麦土壤微生物多样性, 苜蓿茬后第2年小麦微生物多样性最丰富; 粮草短周期轮作则有利于提高小麦土壤真菌多样性, 红豆草茬后第2年小麦真菌多样性最丰富。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。

参考文献

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2009

0.0

0.99

... 土壤微生物是使土壤具有生命力的主要成分, 与土壤肥力和土壤健康有着密切关系, 在土壤形成与发育、物质转化和能量传递等过程中发挥着重要作用, 是评价土壤肥力的重要指标之一^[1] ...

2008

0.0

1.979

林先贵, 胡君利. 土壤微生物多样性的科学内涵及其生态服务功能. 土壤学报, 2008, 45: 892-900

Lin X

, Hu J

Scientific connotation and ecological service function of soil microbial diversity.

Acta Pedol Sin, 2008, 45: 892-900 (in Chinese with English abstract)

Soil microbial diversity is an indicator of species and genes of microbes in the soil ecosystem and their competitive interactions with the environment. The current study in this field is focused mainly on species diversity,genetic diversity,structural diversity and functional diversity. Although the study on soil microbial diversity as a global topic has been carried on forten remaining years,it has displayed features like huge variety of objects,magnitude of contents, broadness in level and novelty in method. However,molecular biological technologies lead and reflect progresses of the research and development of soil microbial ecology. Nowadays,how to improve soil microorganism culturing methods,how to intensify analysis and application of soil metagenomics,how to link soil microbial diversity with service function,and how to expose effect of microhabitat structure on soil microbial diversity,are the four key scientific issues. The basic value of the ecological service functions of soil microbial diversity,of course,lies in decomposing organic matter,recycling nutrients and regulating ecological safety. The study of soil microbial diversity in the years to come should center on its relationship with biological processes and ecological service functions,and establishment of an index system and a methodological system for the study on soil microbial diversity,and elucidation of biological processes of the effect of human activities on soil microbial diversity and its ecological service functions.

土壤微生物多样性是指土壤生态系统中所有的微生物种类、它们拥有的基因以及这些微生物与环境之间相互作用的多样化程度,当前研究主要集中在物种多样性、遗传多样性、结构多样性及功能多样性等4个方面。土壤微生物多样性作为全球性研究迄今仅有10余年时间,但已呈现对象广、内容多、水平宽、方法新等特点,特别是分子生物学技术在很大程度上决定并体现了其研究水平和发展进程。然而,如何进一步改进土壤微生物的培养技能、加强土壤宏基因组学分析与应用、耦合土壤微生物多样性与生态功能、揭示土壤微域结构的影响机制是土壤微生物多样性研究的4个关键科学问题。当然,土壤微生物多样性的生态系统服务功能是其根本价值所在,主要包括有机物分解、物质循环和生态安全调控等3个方面。今后,土壤微生物多样性研究应紧紧围绕其与土壤生物过程、生态服务功能三者之间的联系,着重建立土壤微生物多样性的研究指标和方法体系,进而阐明人类生产活动影响土壤微生物多样性及其生态服务功能的土壤生物过程。

... 土壤微生物多样性指土壤生态系统中所有的微生物种类、它们拥有的基因以及这些微生物与环境之间相互作用的多样化程度^[2] ...

1990

0.0

... 土壤微生物多样性的研究方法很多, 但传统的筛选培养方法仅限于对环境中极少部分可培养微生物类群的研究^[3], 常规分子生物学手段, 如PCR-DGGE、RFLP和RAPD等方法亦因操作复杂、实验周期长、成本高、痕量微生物检测困难等因素无法达到深入分析的目的 ...

... 由于人类可培养的微生物数量大约仅占土壤微生物总数1%左右^[3], 我们认为本研究结果更能真实反映种植制度对旱地土壤微生物的影响状况 ...

2012

2011

38.597

0.0

Star

, Nederbragt A

, Jentoft

, Grimholt

, Malmstrøm

, Gregers T

, Rounge T

, Paulsen

, Solbakken M

, Sharma

, Wetten O

, Lanzén

, Winer

, Knight

, Vogel J

, Aken

, Andersen

, Lagesen

, Tooming-Klunderud

, Edvardsen R

, Tina K

, Espelund

, Nepal

, Previti

, Karlsen B

, Moum

, Skage

, Berg P

, Gjøen

, Kuhl

, Thorsen

, Malde

, Reinhardt

, Du

, Johansen S

, Searle

, Lien

, Nilsen

, Jonassen

, Omholt S

, Stenseth N

, Jakobsen K

The genome sequence of Atlantic cod reveals a unique immune system.

Nature, 2011, 477: 207-210

We sequenced the genome of a heterozygous male Atlantic cod (NEAC_001, Supplementary Notes 1 and 2 ), applying a whole-genome shotgun approach to 40× coverage (estimated genome size of 830 megabases (Mb), Supplementary Note 4 and Supplementary Fig. 2 ) using 454 technology ( Supplementary Note 3 ). Two programs (Newbler 6 and Celera 7 , Supplementary Notes 5 and 6 ) produced assemblies with short contigs, yet with scaffolds of comparable size to those of Sanger-sequenced teleost genomes ( Supplementary Note 10 and Supplementary Fig. 8 ). Although fragmentation due to short tandem repeats is difficult to address ( Supplementary Note 7 ), we resolved numerous gaps attributable to heterozygosity ( Supplementary Note 8 ). The assemblies differ in scaffold and contig length ( Table 1 ), although their scaffolds align to a large extent ( Supplementary Note 9 and Supplementary Fig. 7 ). We obtained about one million single nucleotide polymorphisms (SNPs) by mapping 454 and Illumina reads from the sequenced individual to the Newbler assembly ( Supplementary Note 11 ). Both assemblies cover more than 98% of the reads from an extensive transcriptome data set, indicating that the proteome is well represented ( Supplementary Note 13 ). The assemblies are consistent with four independently assembled bacterial artificial chromosome (BAC) insert clones ( Supplementary Note 14 and Supplementary Fig. 9 ), and with the expected insert size of paired BAC-end reads ( Supplementary Note 15 and Supplementary Fig. 10 ).

... 该技术自2006年问世以来, 已广泛应用于植物学、动物学、医学及极端环境微生物研究^[4,5,6] ...

2011

38.597

0.0

The Potato Genemo Sequencing Consortium. Genome sequence and analysis of the tuber crop potato. Nature, 2011, 475: 189-195

Potato ( Solanum tuberosum L.) is the world’s most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop.

... 该技术自2006年问世以来, 已广泛应用于植物学、动物学、医学及极端环境微生物研究^[4,5,6] ...

2013

3.654

0.0

, Nicolaisen

, Larsen

, Ravnskov

Organic fertilization alters the community composition of root associated fungi in Pisum sativum.

Soil Biol Biochem, 2013, 58: 36-41

... 其中454测序技术因序列读长最长, 目前在土壤微生物多样性研究中应用最广泛^[7,8,9,10] ...

2013

3.654

0.0

Navarro-Noya Y

, Gómez-Acata

, Montoya-Ciriaco

, Rojas-Valdez

, Suarez-Arriaga M

, Valenzuela-Encinas

, Jimenez-Bueno

, Verhulst

, Govaerts

, Dendooven

. Relative impacts of tillage, residue management and crop- rotation on soil bacterial communities in a semiarid agroecosystem. Soil Biol Biochem, 2013, 65: 86-95

... 其中454测序技术因序列读长最长, 目前在土壤微生物多样性研究中应用最广泛^[7,8,9,10] ...

... Navarro-Noya等^[8]认为, 小麦-玉米等禾本科作物轮作及连作不会影响其细菌的丰富度和多样性, 但Sul等^[9]和Acosta-Martí ...

2013

3.654

0.0

Sul W

, Asuming-Brempong

, Wang

, Tourlousse D

, Penton R

, Deng

, Rodrigues

J L M

, Adiku

S G K

, Jones

J W

, Zhou

, Cole

J R

, Tiedje

J M

. Tropical agricultural land management influences on soil microbial communities through its effect on soil organic carbon. Soil Biol Biochem, 2013, 65: 33-38

... 其中454测序技术因序列读长最长, 目前在土壤微生物多样性研究中应用最广泛^[7,8,9,10] ...

... Navarro-Noya等^[8]认为, 小麦-玉米等禾本科作物轮作及连作不会影响其细菌的丰富度和多样性, 但Sul等^[9]和Acosta-Martí ...

... Sul等^[9]认为, 有机碳是造成土壤微生物多样性差异的主要因素 ...

2013

3.654

0.0

Poulsen P H

, Al-Soud W

, Bergmark

, Magid

, Hansen L

. Effects of fertilization with urban and agricultural organic wastes in a field trial—prokaryotic diversity investigated by pyrosequencing. Soil Biol Biochem, 2013, 57: 784-793

... 其中454测序技术因序列读长最长, 目前在土壤微生物多样性研究中应用最广泛^[7,8,9,10] ...

1993

0.0

... 黑垆土是我国黄土高原地区形成的主要地带性耕作土壤之一, 广泛分布在渭北、宁南、陇东和陇中、晋西北地区, 集中分布在黄土旱塬区, 一直是黄土高原重要的农业生产和优质农产品基地, 在黄土高原地区粮食安全中具有重要地位^[11] ...

2009

0.0

1.883

危锋, 郝明德. 长期施用氮、磷和有机肥对不同种植体系土壤有效硫累积的影响. 植物营养与肥料学报, 2009, 15: 613-617

Wei

, Hao M

Soil available sulfur accumulation in a long-term N, P and organic fertilizer experiment under the different cropping systems.

Plant Nutr Fert Sci, 2009, 15: 613-617 (in Chinese with English abstract)

以22年定位试验为基础,研究了长期施用氮、磷和有机肥(NPM)对不同种植体系土壤有效硫在剖面上分布与累积状况.结果表明,在60-80 cm土层各处理出现第一个累积峰,累积峰值为粮饲轮作31.3.玉米连作29.2,小麦连作27.9,粮豆轮作25.6,苜蓿连作24.0 mg/kg;在140-180 cm土层各处理又出现有效硫的第二个累积峰,累积峰值为粮饲轮作44.7,粮豆轮作43.1,小麦连作41.0,玉米连作39.7,苜蓿连作36.5 mg/kg.第二累积峰值均大于第一累积峰值.0-200 cm土层有效硫总累积量粮饲轮作高达746.3 kg/hm2,其次为玉米连作640.6,粮豆轮作为638.3,小麦连作为622.4,苜蓿连作最小为557.3 kg/hm2.长期施用磷肥和有机肥是有效硫在土壤中累积的主要因素,有效硫在土壤剖面上有向深层迁移的趋势.不同作物对硫的吸收利用差异和不同种植方式对有效硫的累积与分布产牛影响.

... 许多学者大量研究了黄土旱作区黑垆土上施肥和种植制度对土壤养分积累、作物产量和品质效应、水分利用效率及土壤环境效应等方面的影响^{[12,13,14,15,16,17]}, 但结合现代分子生物学手段对其土壤微生物多样性的研究尚属空白 ...

2007

0.0

陈磊, 郝明德, 张少民, 高长青. 黄土高原旱地施肥对小麦与苜蓿土壤水分养分含量的影响. 草地学报, 2007, 15: 371-375

Chen

, Hao M

, Zhang S

, Gao C

Effect of long-term fertilization on the soil water and nutrient contents of rainfed wheat and alfalfa land s in Loess Plateau.

Acta Agrestia Sin, 2007, 15: 371-375 (in Chinese with English abstract)

The effects of Long-term fertilization on soil water and nutrient contents of continuous wheat or continuous alfalfa lands under rainfed conditions were studied based on the data of 20-year experiment since 1984. The results show that P application alone didn’t result in the significant increase of wheat yield,but significantly increased the alfalfa yield by 183.5%.The soil water content of alfalfa lands was low and the wheat lands were also found the same trend of getting drier.Long-term P application significantly increased P availability of the soils;soil total P and soil available P of alfalfa lands increased by 13.6% and 83.8% and those of wheat lands increased by 22.9% and 367.9%,respectively.Alfalfa planting had a significant effect on soil fertility,the total N and available N of alfalfa lands increased,but decreased for wheat lands.The application of balanced N-P fertilizer and manure supplied the demanded nutrients to plants and greatly increased the crop yield and soil fertility.

1. Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling, Shaanxi Province 712100, China; 2. Changwu Agricultural Technology Center, Changwu County, Shaanxi Province 713600, China

研究黄土高原旱地小麦(Wheat)和苜蓿(Alfalfa)连作20年后的产量、土壤水分及养分的变化。结果表明:磷肥对小麦增产不显著,而对苜蓿则增产显著(增产183.5%);施肥可以延缓苜蓿的衰老,提高产草量;苜蓿地土壤干燥化明显,施用氮磷有机肥后小麦地也出现干燥化趋势;长期施磷肥有利于改善土壤磷素有效性,其中苜蓿地全磷和有效磷含量,增加了13.6%和83.8%,小麦土壤增加了22.9%和367.9%;苜蓿土壤全氮和速效氮均呈增加,而小麦地则减少;平衡施用氮磷有机肥能培肥土壤,使产量大幅度提高;在该区采取合理的轮作方式和培肥措施能有效地改善农田生态系统的生产力。

... 2%, 与陈磊等^[13]研究结果一致, 这将有利于提高土壤微生物多样性 ...

... 但连续种植苜蓿会加速土壤水分^[13,16]和磷素的耗竭^[28], 降低土壤微生物多样性 ...

... 连续种植小麦后, 虽然有助于改善旱地土壤水分状况^[13,16], 但土壤有效养分含量减少, 会对部分微生物生长产生抑制作用 ...

... 在施氮磷化肥条件下, 粮草长周期轮作中细菌和真菌多样性均大致表现出先降低后增加的趋势, 第4年苜蓿茬及种植苜蓿后第1年小麦茬土壤微生物多样性较低, 可能仍与连续种植苜蓿后土壤干燥化加剧^[13,16], 不利于微生物生长繁殖有关 ...

... 苜蓿茬后种植小麦, 土壤水分逐渐恢复^[13,16], 至第2年小麦茬土壤微生物多样性最高, 且明显高于连作小麦, 此时最有利于发挥土壤潜在肥力, 协调植物养分供应, 提高作物产量和品质^[14,15] ...

2007

0.0

1.883

樊虎玲, 郝明德, 李志西. 黄土高原旱地小麦-苜蓿轮作对小麦品质和子粒氨基酸含量的影响. 植物营养与肥料学报, 2007, 13: 262-266

Fan H

, Hao M

, Li Z

Effect of alfalfa-wheat rotation system on the quality and amino acid content of wheat in dryland of Loess Plateau.

Plant Nutr Fert Sci, 2007, 13: 262-266 (in Chinese with English abstract)

Based on a 20-years long-term fertilization experiment with fixed site on continuous wheat in Changwu,Shaanxi Province,the effect of long-term fertilizer application on the soil fertility and wheat nutrient uptake was studied.Results showed that rational use of chemical fertilizer not only increased wheat yield,but also increased N,P and K content in wheat to some extent.Single applying chemical fertilizer increased N,P and K uptake in wheat and straw,but the combination of N and P had a more pronounced effect,which increased by 143.3%,186.4% and 171.6% in N,P and K uptake in seeds,respectively.The combination of N,P and manure improved soil N,P and K content,increased nutrient content in grain and improved grain quality.Soil organic matter,total phosphorus,available phosphorus and available potassium were increased,while total N and available N were decreased in single phosphorus treatment.The combined application of N,P and manure made the soil organic matter,total N,total P,available N,available P,available potassium increased by 69.0%,57.5%,54.8%,73.1%,830.5% and 119.1%,respectively.It indicated that applying manure could increase soil fertility.

1 College of Resource and Environment Science;Northwest Sci-Tech University of Agriculture and Forest;Yangling 712100;China;2 Institute of Soil and Water Conservation;CAS and MOW;Yangling 712100;China

通过定位试验研究了长期施肥对小麦子粒、秸杆养分吸收和土壤肥力变化的影响。结果表明,合理施用化肥不但增产幅度大,还在一定程度上增加了小麦N、P、K养分浓度。单施氮、单施磷肥能增加小麦子粒和秸杆的N、P、K吸收量,氮磷肥配施增加效果显著,其子粒吸收N、P、K增加了153.2%、157.1%和162.4%。施有机肥能补充土壤中的N、P、K含量,增加小麦子粒中的养分含量,使品质有大幅度的提高。单施磷肥的土壤有机质、全P、速效P含量增加,土壤全N、速效N含量减少,N素亏缺。氮磷肥配施加剧了土壤K的亏缺。有机肥培肥作用显著,有机肥和氮、磷肥配施的土壤有机质、全N、全P、速效N、速效P和速效K分别增加了69.0%5、7.5%、54.8%、73.1%、830.5%和119.1%。

2013

0.0

1.742

蔡艳, 郝明德. 长期轮作对黄土高原旱地小麦籽粒蛋白质营养品质的影响. 应用生态学报, 2013, 24: 1354-1360

Cai

, Hao M

Effects of long-term rotation on the nutritional quality of wheat grain protein on dryland of Loess Plateau, Northwest China.

Chin J Appl Ecol, 2013, 24: 1354-1360(in Chinese with English abstract)

A long-term experiment was conducted on the dryland of Loess Plateau to study the effects of three typical rotation systems, including wheat-sainfoin rotation, wheatpea rotation, and wheat-maize rotation, on the nutritional quality of wheat grain protein. Rotation system and the cropping years of rotated plants affected the nutritional quality of wheat grain protein in varying degrees. As compared with continuous wheat cropping, wheat-sainfoin rotation made the nutritional quality of wheat grain protein relatively stable, and the essential amino acid content, amino acid score, amino acid ratio coefficient, chemical score, and amino acid index of the protein all relatively high, being able to be adopted as a cropping system to product high quality protein wheat in Loess Plateau. Under wheat-pea rotation, the nutritional quality of wheat grain protein after 1year pea cropping was relatively high, but the essential amino acid content of wheat grain protein after 2-year- pea cropping was relatively low, and several essential amino acid scores and chemical score of the grain protein were lower than those under continuous wheat cropping. Furthermore, the essential amino acid index was 12.2% lower than that under continuous wheat cropping. Therefore, wheat-pea rotation showed a relatively low nutritional quality of wheat grain protein. Under wheat-maize rotation, the nutritional quality of wheat grain protein was also relatively stable, but the crude protein and essential amino acid contents and amino acid balance level were lower than those under continuous wheat cropping.

( 1 College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, Shaanxi, China;  2 Institute of Soil and Water Conservation, Northwest A&F University, Yangling 712100, Shaanxi China;  3 College of Resources and Environment, Sichuan Agricultural University, Chengdu 611130, China)

在长期定位试验条件下，研究了黄土高原旱地粮草（小麦-红豆草）、粮豆（小麦-豌豆）和粮饲（小麦-玉米）3种典型轮作制度对小麦籽粒蛋白质营养品质的影响.结果表明：轮作制度及茬口年限可对黄土高原旱地小麦籽粒蛋白质营养品质产生不同程度的影响.与连作小麦相比，实行小麦-红豆草轮作后，小麦籽粒蛋白质营养品质较稳定，且其籽粒蛋白质必需氨基酸含量、氨基酸评分、氨基酸比值系数、化学评分和氨基酸指数均较高，可作为黄土高原生产优质蛋白小麦的轮作制度；小麦与豌豆进行轮作，豌豆茬后1年小麦籽粒蛋白质营养品质较高，但豌豆茬后2年小麦籽粒蛋白质必需氨基酸含量较低，且多种必需氨基酸评分和化学评分低于连作小麦，必需氨基酸指数比连作小麦低12.2%，营养品质较低；虽然粮饲轮作小麦籽粒蛋白质营养品质较稳定，但其粗蛋白、必需氨基酸含量及氨基酸平衡程度均低于连作小麦.

2010

0.0

1.299

李巍, 郝明德, 王学春. 黄土高原沟壑区不同种植系统土壤水分消耗和恢复. 农业工程学报, 2010, 26(3): 99-105

, Hao M

, Wang X

Depletion and restoration of soil water in different cultivating systems in Gully Region of Loess Plateau.

Trans CSAE, 2010, 26(3): 99-105 (in Chinese with English abstract)

黄土高原的土壤干燥化是影响粮食生产可持续发展的关键因素之一,研究土壤干层形成和恢复对该地区粮食生产的可持续发展具有重要意义.该文通过对长期定位观测数据的分析,探讨了不同种植体系和降水年型对土壤水分消耗和恢复的影响.结果表明:与裸地相比,玉米地土壤水分减少最多的月份大多出现在8月或9月,小麦地主要出现在5、6、7月3个月,苜蓿地主要出现在6月或8月;1985-1990年的6 a间,玉米、小麦和苜蓿地生育期内0～2 m土层土壤水分相对裸地平均分别减少了32.52、60.27和86.47 mm.旱作玉米地的土壤水分在平水年型可以得到完全恢复;旱作小麦地在丰水年型或者遇到强降雨的月份土壤水分在0～3 m范围内可以得到恢复,但较难恢复到试验初期的土壤水分.旱作苜蓿地0～3 m范围的土层在降雨较多的年份能够得到一定程度的恢复,6～10 m的土壤水分很难恢复. Abstract： Soil desiccation is one of the key factors to influence the sustainable development of crop production on the Loess Plateau of China. So it has a great significance for the sustainable development of crop production to research the form and recovery of the dry layer of soil. In this article, depletion and restoration of soil water in different cultivating systems were analyzed based on the long-term experiment data at Changwu Agriculture Experimental Station. The results showed that comparing with bare land, the time in which maximal reduction of soil water occurred was August or September in maize land; May, June or July in wheat land; June or August in alfalfa land. In the growth period, from 1985 to 1990, the average soil water in 0-2 m soil layer of maize land, wheat land and alfalfa land decreased by 32.52, 60.27 and 86.47 mm compared with that of bare land. The restoration of soil water in different cultivating systems was different. Soil water of entire dry layer in maize land was thoroughly restored in high precipitation years. Soil water of part dry layer (0-3 m) was restored in wheat land in high precipitation years or in heavy rainfall months, but it was relatively hard to restore to its original level. Soil water in alfalfa land restored to a certain extent in the 0-3 m soil layer in rainy years but hard to restore in the 6-10 m soil layer.

... nez等^[25]分别发现, 冬季栽培豌豆及采取棉花-小麦-玉米轮作比连作更有利于提高土壤细菌多样性, 吴凤芝等^[26]也证明黄瓜与小麦和大豆轮作可显著提高土壤微生物多样性, 说明不同种植制度使土壤温度、湿度等环境因素及土壤微生物可利用的营养发生了不同变化^[16,27], 对其中微生物多样性的影响程度也不完全相同 ...

... 但连续种植苜蓿会加速土壤水分^[13,16]和磷素的耗竭^[28], 降低土壤微生物多样性 ...

... 连续种植小麦后, 虽然有助于改善旱地土壤水分状况^[13,16], 但土壤有效养分含量减少, 会对部分微生物生长产生抑制作用 ...

2009

0.0

高会议, 郭胜利, 刘文兆, 车升国. 黄土旱塬区冬小麦不同施肥处理的土壤呼吸及土壤碳动态. 生态学报, 2009, 29: 2551-2558

Gao H

, Guo S

, Liu W

, Che S

Soil respiration and carbon fractions in winter wheat cropping system under fertilization practices in arid-highland of the Loess Plateau.

Acta Ecol Sin, 2009, 29: 2551-2558 (in Chinese with English abstract)

依据黄土旱塬区黑垆土上中国科学院长武站长期定位试验 (始于1984年),于2008年3月到6月,测定了冬小麦连作系统中返青期、拔节期、抽穗期、灌浆期和收获期土壤呼吸日变化、生育期变化以及土壤可溶性有机碳(Dissolved organic C, DOC)和微生物量碳(Soil microbial biomass C, MBC),研究了施肥措施对土壤呼吸、DOC和MBC的影响以及土壤呼吸与碳组分之间的关系.研究涉及6个处理:休闲地(F)、不施肥(CK)、有机肥(M)、氮肥(N)、氮磷肥(NP)和氮磷有机肥(NPM).结果表明,冬小麦连作系统中土壤呼吸的日变化格局呈单峰曲线,最高值出现在12:00左右(拔节期)和14:30左右(成熟期),最小值出现在0:00～3:00之间或6:00左右;冬小麦土壤呼吸速率拔节期最高,其次是灌浆后期,抽穗期最低;不同施肥条件下,各生育期土壤呼吸速率大小顺序:NPM＞M＞NP＞N＞CK＞F.土壤水分亏缺是导致抽穗期和灌浆期土壤呼吸速率降低的重要原因.各施肥处理DOC含量高低顺序为灌浆期＞抽穗期＞成熟期＞返青期＞拔节期;除M,NPM处理MBC含量拔节期＞灌浆期外,各施肥处理MBC含量高低顺序为成熟期＞抽穗期＞灌浆期＞拔节期＞返青期.同一处理不同生育期土壤呼吸速率与DOC,MBC的相关性较低,但同生育期不同施肥处理土壤呼吸与土壤有机碳组分间存在显著的相关性.以F处理土壤呼吸为基础,估算CK、N和NP处理生育期根系对土壤呼吸的平均贡献率依次为36%、45%和54%.

1991

0.0

... 将纯化后的基因组DNA作为PCR的模板, 采用细菌16S rRNA V1~V3区具有特征性的引物对27F和553R扩增^[18], 其序列分别为27F (5′ ...

1990

0.0

... 采用真菌18S rRNA具有特征性的引物对ITS1和ITS4扩增^[19,20], 其序列分别为ITS1 (5′ ...

1993

6.275

0.0

Gardes

, Bruns T

ITS primers with enhanced specificity for Basidiomycetes—Application to the identification of mycorrhizae and rusts.

Mol Ecol, 1993, 2: 113-118

Department of Plant Pathology, University of California, Berkeley, CA 94720, USA

> We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a ‘universal’ primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the ‘universal’ ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

... 采用真菌18S rRNA具有特征性的引物对ITS1和ITS4扩增^[19,20], 其序列分别为ITS1 (5′ ...

2009

0.0

... 4 生物信息分析方法使用mothur软件完成^[21] ...

2007

0.0

... de/)(silva SSU111版)中的aligned (16S/18S, SSU)核糖体序列数据库比对, 比对方法为kmer searching^[22] ...

2012

3.654

0.0

Russo S

, Legge

, Weber K

, Brodie E

, Goldfarb K

, Benson A

, Tan

Bacterial community structure of contrasting soils underlying Bornean rain forests: inferences from microarray and next-generation sequencing methods.

Soil Biol Biochem, 2012, 55: 48-59

... 3 讨论土壤微生物多样性与表层植被变化有很大关系, 因为植物凋落物或残留物中碳氮的生化组成及可利用程度和养分含量是影响土壤微生物多样性的主要因素^[23,24] ...

2010

5.756

0.0

Campbell B

, Polson S

, Hanson T

, Mack M

, Schuur E

The effect of nutrient deposition on bacterial communities in Arctic tundra soil.

Environ Microbiol, 2010, 12: 1842-1854

1 College of Earth, Ocean, and Environment, University of Delaware, Lewes, DE 19958, USA. 2 Department of Plant and Soil Sciences and 3 Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA. 4 Department of Botany, University of Florida, Gainesville, FL 32611, USA. * E-mail bjc@udel.edu ; Tel. (+1) 302 645 4304; Fax (+1) 302 645 4007.

> The microbial communities of high-latitude ecosystems are expected to experience rapid changes over the next century due to climate warming and increased deposition of reactive nitrogen, changes that will likely affect microbial community structure and function. In moist acidic tundra (MAT) soils on the North Slope of the Brooks Range, Alaska, substantial losses of C and N were previously observed after long-term nutrient additions. To analyse the role of microbial communities in these losses, we utilized 16S rRNA gene tag pyrosequencing coupled with community-level physiological profiling to describe changes in MAT bacterial communities after short- and long-term nutrient fertilization in four sets of paired control and fertilized MAT soil samples. Bacterial diversity was lower in long-term fertilized plots. The Acidobacteria were one of the most abundant phyla in all soils and distinct differences were noted in the distributions of Acidobacteria subgroups between mineral and organic soil layers that were also affected by fertilization. In addition, Alpha - and Gammaproteobacteria were more abundant in long-term fertilized samples compared with control soils. The dramatic increase in sequences within the Gammaproteobacteria identified as Dyella spp. (order Xanthomonadales ) in the long-term fertilized samples was confirmed by quantitative PCR (qPCR) in several samples. Long-term fertilization was also correlated with shifts in the utilization of specific substrates by microbes present in the soils. The combined data indicate that long-term fertilization resulted in a significant change in microbial community structure and function linked to changes in carbon and nitrogen availability and shifts in above-ground plant communities.

2008

3.654

0.0

Acosta-Martínez

, Dowd

, Sun

, Allen

Tag-encoded pyrosequencing analysis of bacterial diversity in a single soil type as affected by management and land use.

Soil Biol Biochem, 2008, 40: 2762-2770

Abstract Impacts of management and land use on soil bacterial diversity have not been well documented. Here we present the application of the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) diversity method, which will promote studies in soil microbiomes. Using this modified FLX pyrosequencing approach we evaluated bacterial diversity of a soil (Pullman soil; fine, mixed, thermic Torrertic Paleustolls) with 38% clay and 34% sand (0–5 cm) under four systems. Two non-disturbed grass systems were evaluated including a pasture monoculture ( Bothriochloa bladhii (Retz) S.T. Blake) [P] and a diverse mixture of grasses in the Conservation Reserve Program (CRP). Two agricultural systems were evaluated including a cotton ( Gossypium hirsutum L.) -winter wheat ( Triticum aestivum L.)-corn ( Zea mays L.) rotation [Ct–W–Cr] and the typical practice of the region, which is continuous monoculture cotton (Ct–Ct). Differences due to land use and management were observed in soil microbial biomass C (CRP > P = Ct–W–Cr > Ct–Ct). Using three estimators of diversity, the maximum number of unique sequences operational taxonomic units (OTU; roughly corresponding to the species level) never exceeded 4500 in these soils at the 3% dissimilarity level. The following trend was found using the most common estimators of bacterial diversity: Ct–W–Cr > P = CRP > Ct–Ct. Predominant phyla in this soil were Actinobacteria , Bacteriodetes and Fermicutes . Bacteriodetes were more predominant in soil under agricultural systems (Ct–W–Cr and Ct–Ct) compared to the same soil under non-disturbed grass systems (P and CRP). The opposite trend was found for the Actinobacteria , which were more predominant under non-disturbed grass systems (P and CRP). Higher G− bacteria and lower G+ bacteria were found under Ct–W–Cr rotation and highest abundance of actinomycetes under CRP. The bTEFAP technique proved to be a powerful method to characterize the bacterial diversity of the soil studied under different management and land use in terms not only on the presence or absence, but also in terms of distribution.

2007

0.0

1.194

吴凤芝, 王学征. 黄瓜与小麦和大豆轮作对土壤微生物群落物种多样性的影响. 园艺学报, 2007, 34: 1543-1546

Wu F

, Wang X

Effect of soybean-cucumber and wheat- cucumber rotation on soil microbial community species diversity.

Acta Hortic Sin, 2007, 34: 1543-1546 (in Chinese with English abstract)

Effects of soybean-cucumber and wheat-cucumber rotation on soil microbial community DNA sequence diversity were evaluated using random amplified polymorphic DNA (RAPD) method. The results showed that grain-cucumber rotation significantly increased the diversity index, richness index and evenness index of soil microbial community, and the soybean-cucumber rotation was more effective than wheat-cucumber rotation in term of the microbial community. Soil microbial community DNA sequence similarity coefficient between the two rotation soils was 0.5678; However the similarity coefficients between the two rotation soils and control soil were 0.3465 and 0.3124, respectively. Cucumber yields in two rotation soils were also higher than that in control soil significantly. This may imply that rotation can improve soil microbial ecology.

(Horticultral College, Northeast Agricultral University, Harbin 150030, China)

采用黄瓜与小麦和大豆轮作的方式，研究轮作对黄瓜土壤微生物群落DNA序列多样性的影响。结果表明，黄瓜与小麦和大豆轮作显著提高了土壤微生物多样性指数、丰富度指数和均匀度指数，两种轮作土壤微生物群落DNA序列相似程度高达0.5678，而与对照土壤间的相似程度分别为0.3465和0.3124。两种轮作处理的黄瓜产量也显著高于对照（ P ＜0.05）。说明轮作改善了土壤的微生态环境。

2000

0.0

0.916

... 长期粮豆轮作不仅可丰富土壤有机质和氮素^[27], 还可利用轮作优势恢复旱地土壤水分, 故可明显提高旱地土壤细菌多样性 ...

... 种植红豆草更有利于土壤有机质累积^[27], 故其后细菌、真菌多样性大致呈现出增加的趋势, 至红豆草茬后第2年小麦土壤微生物多样性达到最高 ...

2008

0.0

1.06

... 但连续种植苜蓿会加速土壤水分^[13,16]和磷素的耗竭^[28], 降低土壤微生物多样性 ...

1990

0.0

... 粮豆轮作土壤真菌多样性不及作物连作, 可能与低营养胁迫下微生物之间竞争有限的营养有很大关系^[29] ...

2003

0.0

0.66

樊军, 郝明德. 长期轮作与施肥对土壤主要微生物类群的影响. 水土保持研究, 2003, 10(1): 88-89

Fan

, Hao M

Effects of long-term rotations and fertilization on soil microflora.

Res Soil Water Conserv, 2003, 10(1): 88-89 (in Chinese with English abstract)

根据长期田间试验,对施肥、作物、种植方式等对土壤细菌、真菌与放线菌含量的影响进行分析.结果显示:施肥,特别是施有机肥明显提高土壤微生物数量,玉米、苜蓿与冬小麦连作不同施肥土壤细菌数量相对休闲地变幅在-197.04×105～+187.07×105个/g土,真菌在-6.08×103～+36.57×103个/g土,放线菌在-47.21×104～+301.4×104个/g土.轮作系统不同施肥处理真菌与放线菌数量高于休闲地.在施氮磷化肥条件下不同作物轮作,苜蓿与糜子对细菌有相对抑制作用,对真菌与放线菌有促进作用,红豆草抑制放线菌促进细菌,豌豆对三类微生物均有抑制作用.

... 樊军等^[30]采用稀释平板法测定种植制度对旱地土壤微生物菌群的影响, 发现低营养条件下土壤可培养微生物表现为小麦连作> ...