青格尔^{1, 2,},
于晓芳^{1, 2,,},
高聚林^{1, 2,,},
王志刚^{1, 2},
胡万吉¹,
闹干朝鲁^{2, 3},
王振^{2, 3},
胡树平^{2, 4},
孙继颖^{1, 2},
屈佳伟^{1, 2}
1.内蒙古农业大学农学院 呼和浩特 010019
2.内蒙古自治区作物栽培与遗传改良重点实验室 呼和浩特 010019
3.内蒙古农业大学园艺与植保学院 呼和浩特 010019
4.内蒙古农业大学职业技术学院 包头 014100
基金项目: 国家自然科学基金项目31760353
内蒙古自然基金项目2020MS03086
内蒙古自然基金项目2018ZD02
国家重点研发计划项目2017YFD0300804
国家玉米产业技术体系项目CARS-02-63
农业部华北黄土高原地区作物栽培科学观测实验站25204120
内蒙古农业大学高层次人才引进科研启动项目NDYB2016-15

详细信息

作者简介:青格尔, 主要从事玉米生理生态研究。E-mail:qinggeer001@163.com

通讯作者:于晓芳, 主要从事作物生理生态研究, E-mail:yuxiaofang75@163.com
高聚林, 主要从事作物生理生态研究, E-mail:nmgaojulin@163.com

中图分类号:S145.6

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出版历程

收稿日期:2020-02-26
录用日期:2020-06-21
刊出日期:2020-11-01

Straw degradation ability and composition of microbial consortium for corn straw decomposition at low temperature

Qing geer^{1, 2,},
YU Xiaofang^{1, 2,,},
GAO Julin^{1, 2,,},
WANG Zhigang^{1, 2},
HU Wanji¹,
Naogan chaolu^{2, 3},
WANG Zhen^{2, 3},
HU Shuping^{2, 4},
SUN Jiying^{1, 2},
QU Jiawei^{1, 2}
1. Agricultural College, Inner Mongolia Agricultural University, Hohhot 010019, China
2. Key Laboratory of Crop Cultivation and Genetic Improvement in Inner Mongolia Autonomous Region, Hohhot 010019, China
3. Horticulture and Plant Protection College, Inner Mongolia Agricultural University, Hohhot 010019, China
4. Vocational and Technical College, Inner Mongolia Agricultural University, Baotou 014100, China
Funds: National Natural Science Foundation of China31760353
Inner Mongolia Natural Sciences Foundation2020MS03086
Inner Mongolia Natural Sciences Foundation2018ZD02
National Key Research and Development Project of China2017YFD0300804
Earmarked Fund for China Agriculture Research SystemCARS-02-63
Crop Science Observation & Experiment Station in Loess Plateau of North China, Ministry of Agriculture, China25204120
Advanced Talented Scholars of Inner Mongolia Agricultural University, ChinaNDYB2016-15

More Information

Corresponding author:YU Xiaofang, E-mail:yuxiaofang75@163.com;GAO Julin, E-mail:nmgaojulin@163.com

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摘要
摘要:根据还田秸秆配施尿素的生产实际，对玉米秸秆低温高效降解复合菌系GF-20进行氮源培养基驯化，探明其菌种组成和功能多样性及其与菌源菌种结构差异，完善复合菌系筛选方法，促进其开发利用。本文以低温高效降解复合菌系GF-20为研究对象，在硫酸铵和尿素不同配比下连续继代培养10代，获得不同氮源菌系（硫酸铵处理N1，硫酸铵和尿素混合处理N2-N5，尿素处理N6），测定其玉米秸秆降解率，评价复合菌系秸秆降解效率；采用MiSeq高通量测序对菌源土壤样品及不同氮源下继代培养的复合菌系菌种组成和功能多样性进行研究。结果显示N2处理玉米秸秆降解率显著高于其他处理；菌源土壤的Alpha多样性指数显著高于继代培养后的复合菌系，不同处理间N2处理显著高于其他氮处理；菌源和复合菌间以及不同氮处理间菌种组成具有显著差异，N2处理菌种组成多样性较高，菌群结构更加丰富、均匀，且碳水化合物的代谢通路丰度较高。菌源经限制性继代筛选后得到了参与玉米秸秆降解过程的功能菌，能有效提高秸秆降解率，其在硫酸铵和尿素氮源为0.16%+0.04%的条件下，菌系的秸秆降解效率较高，这为复合菌的生产实际开发利用提供了理论依据。
关键词:菌源/
复合菌系/
氮源/
高通量测序/
菌种组成多样性/
玉米秸秆降解
Abstract:It is common practice to return field straw with urea to accelerate decomposition. To improve the microbial screening methodology and investigate the microbes responsible for decomposition of corn straw, nitrogen acclimatization of the microbial consortium GF-20 (GF-20) was performed. Compositional differences in the cultured microbial consortium and the microbe source were also evaluated. GF-20 was cultured until the 10^th generation in variable nitrogen conditions[ammonium sulfate (N1), mixtures of ammonium sulfate and urea (N2-N5), and urea (N6)]. The corn straw decomposition ratio was determined to estimate the activity of the composite microbial system, and the composition diversity and function were analyzed by MiSeq high-throughput sequencing. The results showed that the N2 degradation rate was significantly higher than the other treatments. The bacterial source alpha diversity index (ADI) was significantly higher than the cultured microbial consortium, and the N2 ADI was significantly higher than the other nitrogen treatments. The bacterial composition also significantly differed between the source and consortium, as well as among the nitrogen treatments. The N2 treatment yielded the most diverse bacterial composition, with richer and more uniform flora structures and a higher carbohydrate metabolic pathway activity (which promotes corn straw degradation). Functional microbial strains involved in corn straw degradation were obtained after restrictive sub-generation of the microbial sources, which can accelerate corn straw degradation. The highest corn straw degradation efficiency of microbial consortium was observed with the 0.16% ammonium sulfate + 0.04% of urea nitrogen (N2) treatment. These findings provide a basis for developing microbial consortium used in commercial production decomposition.
Key words:Original microbe source/
Microbial consortium/
Nitrogen resources/
High throughput sequencing/
Composition diversity/
Corn straw degradation

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图1不同氮源处理复合菌系玉米秸秆降解率
N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例为分别0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%、0+0.2%。CK为不加菌对照, 培养条件同N1。不同小写字母表示不同处理间差异显著。
Figure1.Corn straw degradation ratios of microbial consortium under different treatments of nitrogen resources
The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, and 0+0.2%, respectively. CK is non-inoculated microbial consortium (control), and the culture conditions are the same as N1. Different lowercase letters mean sig-nificant differences among different treatments.


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图2菌源样品及不同氮源处理复合菌系Alpha多样性指数
Q为菌源土壤。N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例为分别0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%、0+0.2%。
Figure2.Alpha diversity indexes of source samples and microbial consortium under different treatments of nitrogen resources
Q is original microbe source. The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, and 0+0.2%, respectively.


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图3菌源样品及不同氮源处理复合菌系门(a)和纲(b)水平群落结果及属水平(c)Heatmap图分析
将在所有样本中丰度占比均小于0.02%的物种归为Others。Q为菌源土壤。N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例分别为0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%和0+0.2%。
Figure3.Analysis of community structure at phyla (a) and class (b) level and Heatmap at genus level (c) of source samples and mi-crobial consortium under different treatments of nitrogen resources
Microbes with less than 0.02% abundance in all samples are classified as others. Q is original microbe source. The nitrogen resources of N1, N2, N3, N4, N5, and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16% and 0+0.2%, respectively.


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图4菌源及不同氮源处理复合菌系细菌群落结构分布(属水平)
Q为菌源土壤。N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例分别为0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%、0+0.2%。
Figure4.Bacterial community structure at genus level of source samples and microbial consortium under different treatments of nitrogen resources
Q is original microbe source. The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, 0+0.2%, respectively.


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图5不同氮源处理复合菌系属水平PCoA (a)和Rank Abundance (b)分析
N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例分别为0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%和0+0.2%。
Figure5.PCoA analysis (a) and rank abundance analysis (b) at genus level of microbial consortium under different treatments of ni-trogen resources
The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, and 0+0.2%, respectively.


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图6不同氮源处理复合菌系属水平菌种分布比较的Venn图分析
N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例分别为0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%和0+0.2%。
Figure6.Venn analysis at genus level of microbial consortium under different treatments of nitrogen resources
The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, and 0+0.2%, respectively.


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图7不同氮源处理复合菌系COG功能注释相对丰度
N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例分别为0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%和0+0.2%。
Figure7.Relative abundance of the COG annotations of microbial consortium under different treatments of nitrogen resources
The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, and 0+0.2%, respectively.


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图8不同氮源处理复合菌系KEGG主要代谢途径的相对丰度(> 1.00%)
N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例分别为0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%和0+0.2%。
Figure8.Relative abundances of dominant KEGG pathway (> 1.00%) of microbial consortium under different treatments of nitrogen resources
The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, and 0+0.2%, respectively.


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表1不同氮源处理复合菌系KEGG注释结果的第2类水平统计
Table1.Statistics of the KEGG pathway annotations at the second level of microbial consortium under different treatments of ni-trogen resources

KEGG第二代谢水平注释 KEGG pathway level 2	N1			N2			N3			N4			N5			N6
	RA%	log		RA%	log		RA%	log		RA%	log		RA%	log		RA%	log
氨基酸代谢 Amino acid metabolism	12.23	6.27		12.06	6.25		11.59	6.31		11.60	6.35		12.02	6.30		12.52	6.44
碳水化合物代谢 Carbohydrate metabolism	12.71	6.31		14.11	6.24		13.73	6.26		13.99	6.29		13.53	6.28		13.24	6.41
其他次生代谢产物的生物合成 Biosynthesis of other secondary metabolites	0.90	5.26		0.98	5.21		0.83	5.17		0.85	5.20		0.96	5.18		1.04	5.32
辅助因子和维生素的代谢 Metabolism of cofactors and vitamins	6.63	5.98		6.93	5.87		6.58	5.91		6.59	5.94		6.82	5.87		6.79	6.04
其他氨基酸的代谢 Metabolism of other amino acids	2.51	5.56		2.63	5.53		2.71	5.61		2.75	5.65		2.80	5.62		2.88	5.74
萜类和聚酮化合物的代谢 Metabolism of terpenoids and polyketides	3.52	5.54		3.11	5.54		3.15	5.58		3.01	5.62		3.83	5.62		3.04	5.74
异生物素的生物降解与代谢 Xenobiotics biodegradation and metabolism	4.02	5.69		3.87	5.74		4.01	5.83		4.08	5.89		4.57	5.87		4.67	5.98
聚糖的生物合成与代谢 Glycan biosynthesis and metabolism	2.37	5.73		3.22	5.61		2.45	5.61		2.54	5.63		2.35	5.51		2.55	5.74
脂质代谢 Lipid metabolism	3.70	5.81		3.90	5.80		3.67	5.85		3.69	5.90		3.82	5.87		3.75	6.00
能量代谢 Energy metabolism	6.37	6.01		6.77	5.97		6.62	6.00		6.69	6.04		6.56	6.02		6.68	6.14
核苷酸代谢 Nucleotide metabolism	4.70	5.84		5.07	5.75		4.86	5.76		4.91	5.79		4.78	5.78		4.79	5.90
信号转导 Signal transduction	10.17	5.78		8.95	5.62		9.27	5.77		8.92	5.78		8.54	5.63		9.29	5.83
信号分子与相互作用 Signaling molecules and interaction	0.00	4.39		0.00	4.51		0.00	4.67		0.00	4.72		0.00	4.61		0.00	4.73
膜运输 Membrane transport	10.92	6.34		9.29	6.30		11.88	6.45		11.95	6.49		11.30	6.43		9.62	6.46
运输和分解代谢 Transport and catabolism	0.34	4.76		0.49	4.81		0.32	4.78		0.34	4.84		0.42	4.88		0.38	4.91
细胞生长与死亡 Cell growth and death	1.82	0.00		1.62	0.00		1.58	0.00		1.58	0.00		1.34	0.00		1.90	0.00
细胞群落 Cellular community	0.77	5.06		0.54	5.01		0.68	5.00		0.63	5.07		0.50	4.96		0.63	5.27
细胞能动性 Cell motility	3.23	6.06		2.08	5.81		2.67	5.92		2.47	5.92		1.61	5.72		2.90	6.05
循环系统 Circulatory system	0.01	4.11		0.01	3.94		0.01	4.05		0.01	4.07		0.01	3.93		0.02	4.25
消化系统 Digestive system	0.16	4.28		0.26	4.07		0.12	3.92		0.13	3.88		0.14	3.95		0.20	4.14
内分泌系统 Endocrine system	0.35	4.81		0.34	4.75		0.36	4.77		0.35	4.81		0.47	5.04		0.37	4.93
环境适应性 Environmental adaptation	0.26	4.65		0.24	4.44		0.24	4.39		0.23	4.39		0.20	4.38		0.27	4.69
排泄系统 Excretory system	0.01	3.05		0.02	3.60		0.02	3.65		0.02	3.71		0.03	3.93		0.02	3.58
免疫系统 Immune system	0.04	4.12		0.06	3.97		0.05	3.97		0.05	3.95		0.05	4.06		0.05	4.03
神经系统 Nervous system	0.15	3.92		0.13	4.09		0.13	4.40		0.12	4.44		0.12	4.13		0.12	4.13
感官系统 Sensory system	0.00	0.00		0.00	0.00		0.00	0.00		0.00	0.00		0.00	0.00		0.00	0.00
免疫疾病 Immune disease	0.06	4.09		0.05	3.93		0.06	4.08		0.06	4.07		0.07	4.14		0.05	4.09
传染病 Infectious disease	1.65	5.09		1.57	4.95		2.14	5.05		2.09	5.07		1.56	4.87		1.50	5.18
内分泌和代谢疾病 Endocrine and metabolic disease	0.05	4.17		0.05	4.09		0.05	4.18		0.05	4.21		0.06	4.09		0.05	4.23
神经退行性疾病 Neurodegenerative disease	0.20	4.79		0.21	4.83		0.20	4.89		0.20	4.95		0.17	4.88		0.20	5.10
癌症 Cancer	0.20	4.51		0.18	4.53		0.20	4.58		0.20	4.64		0.19	4.58		0.25	4.74
心血管疾病 Cardiovascular disease	0.00	3.18		0.00	3.63		0.00	3.66		0.00	3.80		0.00	3.63		0.01	4.07
复制和修复 Replication and repair	3.81	6.16		4.31	6.08		3.77	6.10		3.79	6.13		4.31	6.12		3.78	6.25
转录 Transcription	0.18	5.72		0.19	5.65		0.17	5.72		0.17	5.75		0.21	5.67		0.18	5.81
翻译 Translation	3.66	6.02		4.23	5.88		3.66	5.87		3.71	5.89		4.26	5.86		3.91	6.05
折叠、分类和降解 Folding, sorting and degradation	2.16	5.78		2.36	5.63		2.10	5.66		2.10	5.67		2.26	5.62		2.14	5.80
N1、N2、N3、N4、N5和N6的氮源为硫酸铵+尿素, 比例分别为0.2%+0、0.16%+0.04%、0.12%+0.08%、0.08%+0.12%、0.04%+0.16%和0+0.2%。RA%指相对丰度, log指注释数量的log10。The nitrogen resources of N1, N2, N3, N4, N5 and N6 are ammonium sulfate+urea with ratios of 0.2%+0, 0.16%+0.04%, 0.12%+0.08%, 0.08%+0.12%, 0.04%+0.16%, 0+0.2%, and respectively. RA% refers to relative abundance, and log refers to log10 of the number of annotations.

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