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1、 Analysis diversity of soil microorganisms about different plant types from Wanning coastal areas A Dissertation Submitted for the Degree of Master Candidate:ZHONG Shengyun Supervisor:HAO Qingyu Hainan Normal University Haikou, China I 摘 要 海南岛人工海防林主要以木麻黄树种为主,与天然海防林相比,存在天然更新能力差,防护期较短,且受台风影响防护效能差,经营成本
2、高等问题,因此迫切需要对木麻黄海防林进行更新改造。若要对木麻黄海防林进行更新改造,筛选出天然更新能力强,适应海岸生态条件,耐土壤贫瘠等优良特性的海南本土树种是最为重要的一个环节。在对海南本土树种初步筛选的基础上,本文拟通过对不同植物类型土壤理化性质的分析,土壤微生物群落多样性的分析,探讨植物-微生物-土壤三者之间的关系,以便揭示不同植物类型对土壤微生物多样性的影响, 并寻找对土壤微生物多样性影响明显的植物类型,即从土壤微生物与植物类型之间关系进一步筛选海防林林替代树种。该项研究将为海防林替代树种的筛选提供新的研究思路与方法。 研究方法:基于对海南岛海防林替代树种的初步筛选,选择了万宁沿海三个地
3、区共八种植物类型: 马骝角的草海桐 (Scaevola sericea) 、 岭南山竹子 (Garcinia oblongifolia) 、刺葵(Phoenix hanceana) 、蕨类植物+野牡丹+桃金娘(灌草丛;shrub-meadow) ;日月湾的对叶榕(Ficus hispida) 、榄仁树(Terminalia catappa)和南燕湾的白楸(Mallotus paniculatus) 、马鞭草科的越南牡荆(Vitex tripinnata) 。 土样采集分别在旱季和雨季进行,雨季取样选在 2013 年 10 月,旱季取样选在 2014 年 4 月。土壤取样是在选定植物周围距离根部
4、 1m 以内,采集 010cm 和 1020cm 两个土层的土样,然后根据五点混合法混合成一个混合土样。采用常规方法测定土壤物理性质(含水率) 、化学性质(有机质、全氮、全磷、全钾、铵态氮、硝态氮、有效磷、速效钾、pH) 。采 用 BIOLOG 微平板计数法测定分析不同植物类型土壤微生物功能多样性,在此基础之上,进一步应用了最能全面解析土壤微生物群落多样性的宏基因组Miseq 测序方法。主要在门和属水平下分析土壤微生物多样性和丰度指数, 指出不同植物类型土壤微生物群落结构组成的差异性。采用多项指标综合分析不同植物类型的土壤微生态环境,筛选沿海地区海防林潜在的替代树种。 主要研究结果: 1.不同
5、植物类型对土壤理化性质的最高值有影响。 岭南山竹子的土壤有机质含量最高;越南牡荆的全氮和全钾含量最高;对叶榕的全磷和硝态氮含量最高;有效磷、速效钾、铵态氮等含量最高的分别是白楸、刺葵和灌草丛。说明植物类型是导致土壤理化性质差异的因素之一; II 2.根据土壤微生物对单一碳源利用能力和多样性指数较为优越或独特的特性, 综合分析 020cm 不同植物类型土壤微生物功能多样性, 结果表明白楸和对叶榕优势种群对总体碳源利用能力较高,榄仁树和灌草丛对总体碳源利用能力较弱; 3.土壤理化性质和土壤微生物对单一碳源总体利用能力的综合分析显示: 上层土的土壤养分和土壤微生物功能多样性较下层土的高;土层对土壤物
6、理、化学性质的影响因植物类型而存在差异; 不同季节土壤理化性质的变化与植物类型及土壤理化性质本身有关; 4.根据高通量 Mesiq 测序结果,七种植物类型获得有效序列数 273454 条,序列数从大到小排列顺序为:榄仁树,对叶榕,刺葵,白楸,岭南山竹子,越南牡荆,灌草丛;在 0.97 的相似度下共获得 6658 个 OTUs,其中榄仁树最高,白楸最低;根据 OTU 丰度进行的 PCA 分析,榄仁树、对叶榕、刺葵,它们的多样性较为相近,越南牡荆和灌草丛的多样性较为相近,白楸、岭南山竹子与其他植物类型的多样性较远,说明同一地区不同植物类型的群落多样性存在差异。 通过不同植物类型土壤微生物多样性及土
7、壤微生物在各分类水平上的百分比综合分析显示, 日月湾的榄仁树和对叶榕的多样性和丰度最高,因此榄仁树和对叶榕是土壤微生物群落多样性较丰富的树种;且它们蕴含的未知序列较大,所以作为开发特殊微生物的树种的潜力最大。 总之,土壤理化性质、土壤微生物功能多样性、土壤微生物群落多样性都随植物类型的不同有不同的变化。在同一取样区,土壤养分、土壤微生物群落多样性较高的植物类型分别是:日月湾的榄仁树;马骝角的刺葵;南燕湾的越南牡荆。从不同植物类型上分析,土壤微生物群落多样性从高到低的总体排序为:榄仁树、对叶榕、刺葵、岭南山竹子、灌草丛、越南牡荆、白楸。 关键词: 土壤微生物群落多样性; 植物类型; 16SrRN
8、A; BIOLOG; 天然海防林; 海南岛 III Abstract Casuarina equisetifolia is the main tree species of coastal artificial windbreaks in Hainan Island. Compared with coastal natural windbreaks, the natural regeneration capability of Casuarina equisetifolia is difficult, protection periods short, protection effect wo
9、rse, especially after suffered typhoon attack, operating cost high, therefore, it is urgent for coastal Casuarina equisetifolia windbreak to be regenerated and transformed. Native tree species of Hainan, with high natural regeneration ability, adaptation to the coastal ecological conditions and tole
10、rating poor sandy soil should be selected as alternative tree species of Casuarina equisetifolia windbreak. Based on the analysis to the soil physical and chemical properties of different plant types, the relationships among different plant types, soil microbial and soil were further studied to reve
11、al the effects of different plant types on soil microbial diversity. In addition, obvious effects of different plant types on soil microbial diversity were analyzed. The study will provide new research ideas and methods for the selection of alternative tree species of coastal windbreak. Research met
12、hod: eight types of plants in three areas of the coastal natural windbreaks in Wanning were selected. There were mainly Scaevola sericea of Maliu corner, Garcinia oblongifolia, Phoenix hanceana and Shrub-meadow, Ficus hispida of Riyue bay and Terminalia catappa, Mallotus paniculatus of Nanyan bay an
13、d Vitex tripinnata. Soil sample were collected in October 2013(rainy season) and April 2014(dry season),two layers soil samples of 010cm and 1020 were collected respectively around each types of selected plants within the scope of less than 1m, and 5 point mixed sampling method was used to make a so
14、il sample. Soil physical properties (moisture content), and chemical properties (organic matter, total nitrogen, total phosphorus, total potassium, ammonium nitrogen, nitrate nitrogen, available phosphorus, rapidly available potassium and pH) were determined by common methods. BIOLOG micro plate cou
15、nt method was used to analyze the effects of different plant types on the soil microbial functional diversity. Furthermore, the metagenomics-Miseq sequencing method was applied to make the comprehensive analysis to the soil microbial community diversity. Soil microbial diversity and richness index w
16、ere analyzed under the levels of phylum and genera. It turned out that the community structure of soil microbial was different over different plant types. Meanwhile, alternative tree species were selected by using comprehensive analysis to the soil micro ecological environment of different plant types. IV Main results for this study are as follows: 1. Different plant types have an influence on the soil physical and chemical characteristics. The highest content of soil organic matt