生态与农村环境学报 ›› 2023, Vol. 39 ›› Issue (1): 123-135.doi: 10.19741/j.issn.1673-4831.2022.0212

• 研究方法 • 上一篇    

基于高通量测序的连续传代富集土壤可培养菌菌群变化规律研究

文昌丽1,2, 曹伟伟2,3, 唐雪莲1, 赵雯淑1, 贾仲君2,3, 孟磊1   

  1. 1. 海南大学热带作物学院, 海南 海口 570228;
    2. 中国科学院南京土壤研究所土壤与农业可持续发展国家重点实验室, 江苏 南京 210008;
    3. 中国科学院大学, 北京 100049
  • 收稿日期:2022-03-17 出版日期:2023-01-25 发布日期:2023-01-14
  • 通讯作者: 贾仲君,E-mail: jia@issas.ac.cn;孟磊E-mail: menglei94@sohu.com E-mail:jia@issas.ac.cn;menglei94@sohu.com
  • 作者简介:文昌丽(1995-),女,贵州平塘人,主要从事土壤微生物方面的研究。E-mail:1378903248@qq.com
  • 基金资助:
    中国科学院战略性先导科技专项(A类)(XDA28020203);国家自然科学基金(42067008); 海南省自然科学基金高层次人才项目(320RC493)

High-throughput Sequencing Technology Based Research on the Changes of Culturable Bacteria Communities in Soil during Serial Enrichment

WEN Chang-li1,2, CAO Wei-wei2,3, TANG Xue-lian1, ZHAO Wen-shu1, JIA Zhong-jun2,3, MENG Lei1   

  1. 1. College of Tropical Crops, Hainan University, Haikou 570228, China;
    2. State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China;
    3. University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2022-03-17 Online:2023-01-25 Published:2023-01-14

摘要: 利用高通量测序技术研究连续传代富集过程中,固体和液体牛肉膏蛋白胨可培养土壤细菌多样性的变化规律,量化可培养菌占水稻土本底微生物群落的比例。通过设置常规营养和1/10低营养处理,包括常规牛肉膏蛋白胨固体培养(NA)、液体培养(NB),低营养固体培养(1/10 NA)和低营养液体培养(1/10 NB),开展连续传代富集10次,获得第1、3、5、7、10代细菌培养物并提取DNA,同时直接提取水稻土中所有微生物基因组DNA并对16S rRNA 基因进行高通量测序,分析水稻土可培养菌群落变化规律及其占本底土著微生物的比例。结果表明,水稻土本底微生物多样性Chao指数为4 806,在连续传代培养10次过程中,降幅最高为98.9%,其中,固体和液体可培养微生物Chao指数以第1代为最低,分别为49.7和142.0,低营养1/10固体和液体培养下,Chao指数分别为75.1和531.0。高通量测序16S rRNA基因后发现水稻土本底土著微生物共713属,连续10次传代富集培养过程中,固体和液体常规培养基中分别检测到52属和600属,低营养1/10固体和液体培养下,分别为62属和597属,可培养菌占比最高分别为8.7%和83.7%。连续第1、3、5、7和10次传代培养过程中,固体培养基每代独有微生物属分别为7、2、3、4和3属,液体培养基独有属则分别为5、1、102、44和24属,低营养1/10培养也得到了类似结果。假单胞菌属(Pseudomonas)是水稻土连续10次传代过程中绝对的优势类群,特别在固体培养基中相对丰度变幅范围为97.70%~99.47%,与水稻土本底土壤中相对丰度相比,增幅最高为74倍。低营养1/10固体条件下,随着传代次数的增加,杆菌属(Bacillus)和代尔夫特菌属(Delftia)成为优势类群,在第10代的相对丰度最高分别为18.07%和13.82%。液体连续传代10次过程中,假单胞菌属也是绝对优势类群,但与固体可培养菌相比略下降为45.48%~55.99%,梭菌属(Clostridium)则是第2大优势类群,其相对丰度范围为23.58%~42.40%,而赖氨酸芽孢杆菌属(Lysinibacillus)则保持相对稳定的增长趋势,其相对丰度范围为1.46%~6.74%。相反,低营养1/10液体培养下,梭菌属和赖氨酸芽孢杆菌属随着培养代数增加而急剧降低,假单胞菌属则成为绝对优势菌,而类芽孢杆菌属(Paenibacillus)则呈现随着传代次数增加而增加的趋势。上述结果表明,水稻土本底微生物多样性最多达可培养菌的96.7倍,连续传代富集培养过程中,水稻土可培养微生物Chao指数先增加后降低,固体和液体培养分别获得74和662属,可培养菌占水稻土所有微生物比例分别为10.4%和92.8%。在固体和液体传代培养过程中,假单胞菌属均占据绝对优势地位,液体可培养微生物多样性明显高于固体。低营养1/10固体和液体培养条件下,可培养微生物类群显著增加,第1代出现的部分微生物类群逐渐被某些优势菌替代,表明连续传代富集培养过程中,在不同代际均富集了生理代谢特征具有明显差异的类群,连续传代富集并不会导致种群结构同质化和单一化。

关键词: 微生物可培养法, 连续传代, 固液体培养基, 氮素营养, 高通量测序

Abstract: High-throughput sequencing technology was used to study the changes in the diversity of culturable bacteria in solid and liquid beef extract peptone soil during serial enrichment, and to quantify the proportion of culturable bacteria in the background microbial community of a paddy soil. Serial passages were performed by setting up treatments of conventional nutrient and 1/10 low nutrient treatments, including conventional beef extract peptone solid (NA) and liquid (NB) cultures, as well as low nutrient solid (1/10 NA) and low nutrient liquid (1/10 NB) cultures. The cultures were enriched 10 times to obtain the 1st, 3rd, 5th, 7th, and 10th generation of bacterial cultures and then to extract their DNAs. At the same time, all microbial genomic DNAs in the paddy soil were directly extracted and high-throughput sequencing 16S rRNA gene was used to analyze the change of culturable microorganisms communities in the paddy soil and their proportions to the background indigenous microorganisms. The results show that the background Chao index of the microbial diversity of the paddy soil was 4 806, with a maximum reduction of 98.9% in 10 consecutive generations. Among them, the Chao index of solid and liquid culturable microorganisms was the lowest in the first generation, which were 49.7 and 142, respectively. Under 1/10 solid and 1/10 liquid cultures, the Chao index was 75.1 and 531, respectively. High-throughput sequencing of 16S rRNA genes revealed a total of 713 genera of background indigenous microorganisms in paddy soil. During the enrichment culture process of 10 consecutive passages, 52 and 600 genera were detected in solid and liquid conventional culture, respectively. However, 62 and 597 genera were detected in 1/10 solid and 1/10 liquid cultures, respectively. The highest proportion of culturable bacteria was 8.7% and 83.7%, respectively. During the 1st, 3rd, 5th, 7th and 10th consecutive subcultures, 7, 2, 3, 4 and 3 genera were unique to each passage in solid medium, while the unique genera in liquid culture medium were 5, 1, 102, 44, and 24 genera, respectively, and similar results were observed in low-nutrient (1/10) cultures. Pseudomonas was an absolute dominant group in paddy soil during 10 consecutive passages, especially in solid medium, the relative abundance range was 97.70%-99.47%, which is 74 times higher than that of the background in the paddy soil. Under the low-nutrient 1/10 solids, Bacillus and Delftia became the dominant taxa with the increase of passage times, and the highest relative abundance in the 10th generation was 18.07% and 13.82%, respectively. During the 10 consecutive liquid passages, Pseudomonas was also an absolutely dominant group, but compared with solid culturable bacteria, it decreased slightly to 45.48%-55.99%, while Clostridium was the second most dominant group, with a relative abundance ranged from 23.58% to 42.40%, while the genus Lysinibacillus only observed in the 5th, 7th and 10th generations, and its abundance ranged from 1.46% to 6.74%. On the contrary, under the condition of low nutrient 1/10 NB liquid, Clostridium and Lysinibacillus decreased sharply with the increase of culture generation, Pseudomonas became the absolute dominant bacteria, while Paenibacillus increased with increasing passage number. These results show that the background microbial diversity of paddy soil can be up to 96.7 times of culturable bacteria. During the continuous enrichment culture, the Chao index of culturable microorganisms in paddy soil first increased and then decreased, and 74 and 662 genera were obtained by solid and liquid cultures, respectively. The proportion of solid and liquid cultured bacteria in all microorganisms in paddy soil was 10.4% and 92.8%, respectively. In the process of solid and liquid subculture, Pseudomonas was the dominant group, and the diversity of culturable bacteria in liquid was significantly higher than that in solid culture. Under low-nutrient 1/10 solid and 1/10 liquid culture conditions, the culturable microbial groups increased significantly, and some of the microbial groups that appeared in the first generation were gradually replaced by some dominant bacteria, indicating that in the process of continuous enrichment culture, the average number of bacteria in different generations increased. The groups with obvious differences in physiological and metabolic characteristics were enriched, and serial enrichment did not lead to homogenization and simplification of the population structure.

Key words: culturable method of microorganism, continuous passage, solid liquid medium, nitrogen nutrition, high-throughput sequencing

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