刘岳飞(1989—),男,河南洛阳人,硕士研究生,主要从事设施栽培生理与环境调控方面的研究。E-mail:
以我国南方植椒土壤为研究对象,基于传统和高通量测序技术,研究覆盖不同地膜对土壤微生物生物量、酶活性和细菌多样性的影响。结果发现,覆盖地膜(尤其是以聚乙烯为原料的无色透明地膜、黑色地膜和银色地膜)不仅有助于提高耕作层土壤温度与含水量,而且对减缓土壤pH下降以及促进土壤微生物生物量与酶活性的提高均具有显著效果。从土壤细菌群落组成来看,供试土壤中黄杆菌属(
Soil erosion and declining of soil fertility is becoming a severe problem in vegetable fields in South China. In practice, mulching system has become one of the effective measures for solving this problem. However, the study of the different mulching on soil biological properties and bacterial diversity in vegetable fields were rarely investigated.
Soil samples were collected from pepper fields with different mulching treatments in South China. Soil temperature, water contents, soil microbial biomass, enzyme activity were analyzed by traditional ways and bacterial diversity was studied by using high-throughput sequencing techniques.
The soil temperatures in different layers from the depths of 5 cm, 10 cm, 15 cm and 20 cm were increased 0.05~2.93 ℃, 0.06~1.88 ℃, 0.45~2.16 ℃ and 0.23~2.46 ℃, respectively. Meanwhile, soil water content was increased 13.3%~19.4% under different mulching conditions which compared to control. By contrast, soil pH was also raised from 0.01 to 0.19 under different mulching conditions which compared to CK. In addition, the enzyme activity and microbial biomass were also significantly increased which relative to type of the mulch, i.e. in turn, colorless transparent film, black film and silver film which are all made of polyethylene. Soil bacterial diversity,
The colorless transparent plastic film mulching is the best for increasing soil temperature, water content and enzyme activity. And all the mulching treatments not only can improve the soil physical and chemical characteristic, increase the soil microbial biomass and enzyme activities, but also can accumulate some of the plant growth promoting bacteria (PGPB) in pepper fields. All the findings indicate that instead of degrading soil quality of the pepper fields, the current practice of film mulching will help build up soil fertility and improve soil health
广西是我国最大的秋冬蔬菜生产基地,其中辣椒的种植面积最大[
鉴于此,本文研究覆盖不同种类地膜对我国南方植椒土壤温度、理化性状及生物学特性的影响,旨在评价覆盖地膜对植椒土壤肥力与健康状况的影响,为我国南方地区科学利用地膜提供理论依据和技术支撑。
试验于2016年在广西大学农学院蔬菜生产基地(东经108º18′,北纬22º51′)进行,辣椒品种为“桂椒8号”,于2月下旬育苗,4月中旬定植。试验地土壤类型为赤红壤,试验前土壤pH6.08, 有机质含量7.30 g·kg-1,全氮0.82 g·kg-1,全磷0.51 g·kg-1,全钾14.57 g·kg-1,碱解氮57.60 mg·kg-1,有效磷3.22 mg·kg-1,速效钾82.94 mg·kg-1。
试验设五个处理:不覆膜对照(M1)和覆盖黑色聚乙烯地膜(黑色地膜,M2)、无色透明聚乙烯地膜(无色透明地膜,M3)、银色聚乙烯地膜(银色地膜,M4)、麻类材料地膜(M5),每个处理3个重复,随机排列。每个小区面积30 m2,株距30 cm×30 cm。辣椒定植后,施肥等日常管理按常规方法统一进行。
在辣椒采收期间(8月25日)按“S”型采集耕作层(0~30 cm)土壤样品。每个小区分别采集5~8个点,去除残根等杂物后充分混匀,并用无菌塑料袋装好带回实验室。一部分室内自然风干后过40目筛,用于土壤理化性状测定;另一部分过10目筛后,置于4℃冰箱保存,用于土壤生物学性状分析(其中用于高通量测序分析的样品在采集后按测序样品要求即时处理并包装送检)。
2016年6月1日至8月25日每天早上10:00,采用WQG-16曲管地温表读取不同深度(5、10、15、20 cm)土壤温度。土壤含水量、pH和有机质分别采用称重法、PHS-3C型精密酸度计和重铬酸钾容量法测定。
土壤微生物生物量碳、氮和磷测定采用氯仿熏蒸提取-容量分析法[
总DNA提取与PCR扩增:采用OMEGA土壤提取试剂盒提取总DNA,用Qubit2.0检测DNA浓度,琼脂糖凝胶检测DNA完整性。每个样品3次重复,将同一样品的DNA混合,作为每份土壤样品的总DNA。PCR扩增采用的是16S rRNA基因中V3-V4高变区通用引物341F(CCTACGGGNGGCWGCA)和805R(GACTACHVGGGTATCTAATCC)。扩增条件设置:94℃预变性3 min,94 ℃变性30s,45℃退火20 s,65 ℃延伸30s,5个循环;94 ℃延伸20 s,55℃退火20 s,72 ℃延伸30 s,20个循环。进一步采用匹配Illumina测序接头和标签引物扩增,采用生工琼脂糖回收试剂盒(cat:SK8131)对DNA进行回收。回收产物用Qubit2.0定量,根据测得的DNA浓度将所有样品按照1:1的比例进行混合,用于后续测序。
高通量测序:16S rRNA基因的PCR产物由北京百迈客生物科技有限公司完成测序,测序仪器为Illumina公司的新一代测序仪Hiseq 2500,测序所得结果,采用prinseq软件对reads1、reads2的3端进行质控,截掉质量低的数据,以提高后续序列融合比率。通过Flash软件融合双末端序列,使其形成一条序列。采用prinseq软件对各个样品进行去引物序列、短片段、低复杂度序列、低质量序列。
试验数据采用Excel 2010进行计算处理,采用SPSS 19.0软件进行方差分析,显著性检验采用邓肯(Duncan)法,不同字母表示在
覆盖不同种类地膜对植椒土壤温度、含水量和pH的影响
Effects of mulching on soil temperature, water content, and pH in pepper fields relative to type of the mulching film
处理 |
土壤温度Soil temperature/℃ | 含水量 |
pH | |||
5 cm | 10 cm | 15 cm | 20 cm | |||
注:M1,CK;M2,黑色地膜;M3,无色透明地膜;M4,银色地膜;M5,麻类材料地膜。表中数据为平均值±标准差。同列字母不同表示处理间差异显著( | ||||||
M1 | 30.62±0.90d | 23.97±0.64d | 29.86±0.52d | 29.91±0.24d | 164.5±24.2c | 5.64 ±0.02c |
M2 | 32.55±1.33b | 25.42±1.03b | 31.96±0.92a | 32.14±0.44a | 187.0±2.1b | 5.74±0.01b |
M3 | 33.21±0.36a | 25.85±0.29a | 32.02±0.35a | 32.37±0.28a | 196.7±7.1a | 5.77 ±0.01b |
M4 | 31.80±0.61c | 25.07±0.38c | 31.22±0.39c | 31.44±0.46b | 193.3±8.0a | 5.83 ±0.02a |
M5 | 30.67±0.65d | 24.03±0.50d | 30.31±0.43c | 30.14±0.34c | 16.36±18.5c | 5.65±0.02c |
与对照相比,覆盖除麻类材料外的地膜均显著提高了植椒土壤的含水量,并显著提高了土壤pH,即聚乙烯类材料地膜更有助于保持土壤水分含量和减缓土壤pH下降。
由
覆盖不同种类地膜对植椒土壤微生物生物量及酶活性的影响
Effects of mulching on soil microbial biomass and enzyme activities in pepper fields relative to type of the mulching film
处理 |
微生物生物量碳 |
微生物生物量氮 |
微生物生物量磷 |
β-葡糖苷酶 |
氨肽酶 |
磷酸酶 |
M1 | 359.5±22.6e | 57.6±3.8b | 28.1±5.6b | 0.41±0.12d | 16.26±0.24c | 0.35±0.07d |
M2 | 445.9±15.6d | 69.7±3.9ab | 69.2±1.6a | 1.87±0.11c | 16.36±0.68c | 0.72±0.04b |
M3 | 706.9±39.2b | 73.3 ±8.5a | 60.0±5.6a | 2.74±0.26a | 17.61±0.41a | 1.45±0.20a |
M4 | 761.2±8.1a | 62.6±5.5ab | 65.1±3.7a | 2.14±0.16b | 16.79±0.43b | 0.57±0.01c |
M5 | 498.8±15.5c | 76.1±6.0a | 34.1±2.3b | 0.55±0.03d | 16.33±0.64c | 0.36±0.0d |
测序共获得有效序列348 687条,平均长度为422 bp,测序覆盖率在82.4%~84.8%。根据分类学分析结果,可以得知不同分组(或样本)在属分类水平上的群落结构组成情况(
土壤细菌属分类水平的组成和相对丰度(a)及在OTU水平的维恩图(b)
Composition and relative abundance of the soil bacteria at genus level (a) and Venn diagram of the soil bacteria at OTU level (b)
维恩图可以反映环境样品的相似性及重叠情况,由
微生物群落多样性特征分析中,Chao1、ACE、香农指数越大,说明样品的物种多样性越高[
覆盖不同种类地膜对植椒土壤细菌多样性的影响
Effect of mulching on soil bacterial diversity in pepper fields relative to type of the mulching film
处理 |
Chao1指数 |
ACE指数 |
香农指数 |
覆盖率 |
M1 | 2 054±80c | 2 131±68b | 6.04±0.05c | 0.997 |
M2 | 2 083±79c | 2 084±52c | 6.06±0.04c | 0.997 |
M3 | 2 162±99a | 2 143±72a | 6.52±0.07a | 0.998 |
M4 | 2 141±92b | 2 136±71b | 6.17±0.09b | 0.998 |
M5 | 2 141±89b | 2 128±64b | 6.28±0.03b | 0.998 |
地膜具有调节土温、保持水土、维持土壤结构、防治杂草与病毒病以及防止土壤盐类积累等作用,而且黑色和银色地膜提高土温效果不及透明地膜[
土壤微生物活性与多样性的变化能敏感地反映出土壤生态系统的质量和健康状况[
基于高通量测序技术分析发现,覆盖不同地膜还有利于提高植椒土壤细菌丰富度和多样性指数,尤其以无色透明地膜的提升效果尤为显著,其原因可能与无色透明地膜显著提高土壤温度、含水量和减缓土壤pH下降的作用紧密相关。根据位于北方地区的研究中发现,地膜覆盖显著影响土壤微生物的群落结构,其影响效果主要通过对土壤有机碳、总氮、有效磷、pH以及土壤水分的间接作用实现的[
虽然在97%相似度的聚类分析还未能完全查明土壤细菌遗传多样性,但是基于高通量测序深度基本能够反映该区域细菌群落的种类和结构,完全可以定量比较整个群落组成和多样性的相对差异[
当前南方地区辣椒实际生产中,覆盖不同种类地膜(尤其以聚乙烯为原料的无色透明地膜、黑色地膜和银色地膜)不仅有助于提高辣椒菜园耕作层土壤温度与含水量,而且对减缓南方地区植椒土壤pH的下降、提高土壤微生物生物量以及酶活性具有显著的效果。同时,应用地膜还有助于提高植椒土壤中细菌的丰富度(Chao1)和多样性(Shannon)指数。另一方面,黄杆菌属、溶杆菌属、产碱杆菌属、鞘氨醇单胞菌属和芽胞杆菌属细菌等一些具有生防作用的益生细菌是覆盖地膜植椒土壤中的优势菌属,表明当前南方地区辣椒生产中应用地膜不仅没有导致植椒土壤质量劣化,而且还有助于提高土壤肥力和保持土壤健康。
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