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林业科学 ›› 2018, Vol. 54 ›› Issue (9): 169-176.doi: 10.11707/j.1001-7488.20180919

• 研究简报 • 上一篇    下一篇

杨树人工林土壤微生物群落4种提取方法比较

秦媛1,2, 潘雪玉1, 靳微1, 陈连庆1, 袁志林1   

  1. 1. 中国林业科学研究院亚热带林业研究所 杭州 311400;
    2. 中国科学院遗传与发育生物学研究所 北京 100020
  • 收稿日期:2017-05-30 修回日期:2017-11-22 出版日期:2018-09-25 发布日期:2018-09-10
  • 基金资助:
    中国林业科学研究院中央级公益性科研院所基本科研业务费专项资金(子课题)(重点项目CAFYBB2017ZB001);2016年度留学回国人员科技活动项目择优资助经费和国家自然科学基金优秀青年项目(31722014)。

Comparison of Four Extraction Methods of Soil Microbiome in Poplar Plantation

Qin Yuan1,2, Pan Xueyu1, Jin Wei1, Chen Lianqing1, Yuan Zhilin1   

  1. 1. Research Institute of Subtropical Forestry, CAF Hangzhou 311400;
    2. Institute of Genetics and Developmental Biology, CAS Beijing 100020
  • Received:2017-05-30 Revised:2017-11-22 Online:2018-09-25 Published:2018-09-10

摘要: [目的]综合评价4种土壤微生物细胞提取技术的优劣,旨在为阐明土壤微生物群落对林木生长生理的调控作用以及建立林木根际微生物组工程实验体系提供技术依据。[方法]以杨树人工林非根际土壤为材料,使用超声波、搅拌器2种物理分散方法和焦磷酸钠、吗啉乙磺酸一水合物2种化学分散剂对土样进行预处理,并通过2步离心法获得菌体样品。经微生物群落扩增子高通量测序、BIOLOG生态板和真菌板碳源利用比较、真菌和细菌菌落形成单位计数等分析手段,评价不同提取方法所得菌体样品(包括原始土样)在微生物物种多样性、群落结构特征和群落水平生理代谢等方面的差异。[结果]4种提取方法所得菌体样品的操作分类单元(OTU)数量及多样性指数与原始土样相比均呈现不同程度的降低,其中经焦磷酸钠和搅拌器处理后所得样品的微生物群落多样性和结构组成与原始土样最为接近;可培养菌落总数较高,代谢活性(碳源利用能力)相对较强。相反,超声波处理后细菌物种多样性最低,可培养真菌数量显著低于焦磷酸钠和搅拌器2种处理(P<0.05),且BIOLOG分析结果表明菌群代谢活性较低。吗啉乙磺酸一水合物处理后所得样品菌群结构与原始土样差异较大,且可培养细菌数量显著低于其他处理组(P<0.05)。[结论]4种提取方法对土壤微生物群落结构、组成和代谢活性等方面有不同程度的影响,其中超声波处理和吗啉乙磺酸一水合物溶液处理提取效果较差,采用焦磷酸钠和搅拌器处理能更完整、有效提取土壤菌体细胞并维持菌群较高代谢活力。

关键词: 高通量测序, BIOLOG, 菌落形成单位, 微生物群落水平生理特征, 根际微生物组

Abstract: [Objective] Efficient extraction of soil microbiome is a crucial step for investigating the physiology and ecology of microbial communities as well as interactions between plant and its associated microbiota. In this work, we compared the microbial diversity and metabolic activity of soil microbiome in poplar plantation through four extraction techniques. The aim is to provide the efficient and reliable means that enables extracted samples to represent for the natural soils. This technique can be further applied in tree rhizosphere microbiome engineering.[Method] The bulk soils in poplar plantation were collected. The soil samples were pretreated with two physical dispersing methods of blending and sonication and two kinds of chemical dispersants of sodium pyrophosphate and MES monohydrate, and then the microbial pellets were obtained through two-step centrifugation. Microbial community structure and diversity, viable counts and the community level physiological profiling in the extracted microbial cell samples were evaluated and compared through the integration of 16S rRNA and ITS high-throughput pyrosequencing, BIOLOG analysis (ECO and FF plates) as well as the colony-forming unit (CFU) assay of culturable fungi and bacteria.[Result] Results showed that, compared to the natural soil microbiome, all treatments had slight negative influence on the species diversity (OTUs and diversity index). When soils were treated with sodium pyrophosphate and blending, the diversity and community structure of the extracted samples were relatively closer to natural bulk soils. In addition, the CFU counts and metabolic activity (carbon utilization capacity) were relatively higher than other treatments. On the contrary, with the sonication treatment the diversity of bacterial species was the lowest, and the number of cultivated fungi was significantly lower than that of two treatments with sodium pyrophosphate and blending ( P <0.05). Moreover, the microbial pellet also showed a lower metabolic activity evidenced by the BIOLOG analysis. The community structure of pellet samples extracted with MES monohydrate differed greatly compared to the natural soils, and the number of culturable bacteria was significantly less than other treatments (P <0.05).[Conclusion] The four extraction methods have different effects on the structure, composition and metabolic activity of soil microbial community, in which the sonication and MES monohydrate treatments maintain a relatively low level of microbial diversity and metabolic activity compared to the natural soils. Therefore, we suggest that the sodium pyrophosphate and physical blending, either alone or in combination, will be good alternatives for extracting the soil microbiome efficiently.

Key words: high-throughput sequencing, BIOLOG analysis, colony-forming unit(CFU), community level physiological profiling, rhizosphere microbiome

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