高寒地区根瘤菌拌种对禾/豆混播土壤微生物群落的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-1066

摘要/Abstract

摘要：

拌种根瘤菌是提高植物固氮效率的有效手段之一。本研究探讨了根瘤菌拌种处理对高寒地区典型禾/豆混播中土壤微生物群落结构和多样性的影响，旨在为根瘤菌拌种在牧草生产应用中提供理论依据。以苜蓿‘北林201’、‘川草2号’老芒麦和‘阿坝’垂穗披碱草混播为研究对象，设置根瘤菌拌种和不拌种两个处理。采集根周土壤和根际土壤，基于16S rRNA基因高通量测序技术分析原核生物多样性和群落结构，FAPROTAX对不同处理原核微生物群落功能进行预测，并通过BugBase分析比较不同处理下原核微生物高水平表型的分类和变化。32个土壤样品共检测到8 814个OTU，分属于6个门3 577个属。优势门为放线菌门（Actinobacteria）（26.11%-46.20%）和变形菌门（Proteobacteria）（20.61%-32.91%）。拌根瘤菌改变了牧草土壤中原核生物的群落结构，拌种处理的原核微生物多样性指数（Observed_richness、Shannon index、inverse Simpson index）都显著（P < 0.05）低于不拌种。拌根瘤菌增加了消化作用以及反硝化功能的相对丰度。拌种处理后禾本科根际土壤原核微生物的需氧、厌氧、兼性厌氧、革兰氏阴性、革兰氏阳性的种群增加，而豆科结果与之相反。拌种后，豆科根际的nifH基因显著高于不拌种的丰度。拌根瘤菌降低了禾/豆混播草地土壤原核微生物的α多样性，改变了土壤原核微生物群落的结构组成，增加了消化作用和反硝化作用种群的相对丰度，并提高了nifH基因的丰度。本研究为根瘤菌拌种在禾/豆混播栽培方式提供了科学依据。

关键词: 根瘤菌拌种, nifH基因, 微生物多样性, 微生物群落结构, 功能预测

Abstract:

Rhizobium seed dressing is one of the effective measures to improve the nitrogen fixation efficiency of plants. Our study investigated the effects of rhizobium seed dressing on the structure and diversity of soil microbial community in typical grass-legume mixtures in alpine regions，aiming to provide a theoretical basis for the application in forage production. Taking the mixed sowing of Medicago sativa cv.‘Beilin201’，Elymus sibiricus L.cv.‘Chuancao No.2’，and Elynus nutans Griseb.cv.‘Aba’ as the research object，2 treatments of rhizobium seed dressing and no seed dressing were set. The bulk and rhizosphere soil were collected，and the prokaryotic biodiversity and community structure were analyzed based on 16S rRNA gene high-throughput sequencing technology，the functional prediction was calculated by FAPROTAX tools and the classification and changes of prokaryotes microbial high-level phenotypes were compared by BugBase analysis. A total of 8 814 OTUs were detected from 32 soil samples，belonging to 3 577 genera in 6 phyla，while the dominant phyla were Actinobacteria（26.11%-46.20%）and Proteobacteria（20.61%-32.91%）. Rhizobium seed dressing changed prokaryotic community structure and decreased diversity（Observed_richness，Shannon index，and inverse Simpson index），but increased the abundance of digestion and denitrification taxa. The population belonging to aerobic，anaerobic，facultative anaerobic，gram-negative，and gram-positive prokaryotes in the rhizosphere soil of Gramineae increased after seed dressing，while the results of Legumes were the opposite. After seed dressing，the abundance of nifH gene in the legume rhizosphere significantly increased. In summary，seed dressing reduced the α-diversity of community，changed the structural composition，but increased the relative abundances of digesting and denitrifying populations，and increased the abundance of nifH genes. This study provides a scientific basis for the cultivation of rhizobia seed dressing in the grass-legume mixtures.

Key words: rhizobium with seed dressing, nifH gene, microbial diversity, structure of microbial community, function prediction

颜珲璘, 芦光新, 邓晔, 顾松松, 颜程良, 马坤, 赵阳安, 张海娟, 王英成, 周学丽, 窦声云. 高寒地区根瘤菌拌种对禾/豆混播土壤微生物群落的影响[J]. 生物技术通报, 2022, 38(10): 204-215.

YAN Hui-lin, LU Guang-xin, DENG Ye, GU Song-song, YAN Cheng-liang, MA Kun, ZHAO Yang-an, ZHANG Hai-juan, WANG Ying-cheng, ZHOU Xue-li, DOU Sheng-yun. Effects of Rhizobium Seed Dressing on the Soil Microbial Community of Grass-legume Mixtures in Alpine Regions[J]. Biotechnology Bulletin, 2022, 38(10): 204-215.

图/表 11

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牧草种类 Forage species	混播草地 Mixed pasture	重复数 Repetition	标记 Remark
‘川草2号’老芒麦（Elymus sibiricus L.cv.‘Chuancao No.2’）	苜蓿‘北林201’（拌种） Medicago sativa cv.‘Beilin201’（Rhizobium）	3	A1
‘川草2号’老芒麦（Elymus sibiricus L.cv.‘Chuancao No.2’）	苜蓿‘北林201’Medicago sativa cv.‘Beilin201’	3	D1
‘阿坝’垂穗披碱草（Elynus nutans Griseb.cv.‘Aba’）	苜蓿‘北林201’（拌种）Medicago sativa cv.‘Beilin201’（Rhizobium）	3	A2
‘阿坝’垂穗披碱草（Elynus nutans Griseb.cv.‘Aba’）	苜蓿‘北林201’Medicago sativa cv.‘Beilin201’	3	D2

牧草种类 Forage species	混播草地 Mixed pasture	重复数 Repetition	标记 Remark
‘川草2号’老芒麦（Elymus sibiricus L.cv.‘Chuancao No.2’）	苜蓿‘北林201’（拌种） Medicago sativa cv.‘Beilin201’（Rhizobium）	3	A1
‘川草2号’老芒麦（Elymus sibiricus L.cv.‘Chuancao No.2’）	苜蓿‘北林201’Medicago sativa cv.‘Beilin201’	3	D1
‘阿坝’垂穗披碱草（Elynus nutans Griseb.cv.‘Aba’）	苜蓿‘北林201’（拌种）Medicago sativa cv.‘Beilin201’（Rhizobium）	3	A2
‘阿坝’垂穗披碱草（Elynus nutans Griseb.cv.‘Aba’）	苜蓿‘北林201’Medicago sativa cv.‘Beilin201’	3	D2

环境因子Environmental factors	变量Variables	分组Group
环境因子Environmental factors	变量Variables	DB	DN	HB	HN
理化性质 Soil physical-chemical property	总氮TN/%	1 805.37±293.06	1 814.36±152.19	1 676.88±248.44	1 777.86±137.70
	铵态氮NH₄^-N /（mg·kg^-1）	4.41±1.11	4.22±0.54	4.52±0.94	4.30±1.00
	硝态氮NO₃^-N /（mg·kg^-1）	38.87±2.85	39.97±1.70	41.84±4.35	40.06±2.08
	有机碳TOC/%	2.29±0.30	2.24±0.29	2.23±0.31	2.17±0.28
土壤性质 Soil property	温度MAT/℃	13.49±3.54	11.88±0.18	13.98±4.36	11.87±0.20
	水分SMC/%	9.81±1.06	10.63±1.31	10.63±1.23	10.70±1.45
	电导率EC/（us·cm^-1）	0.06±0.04	0.04±0.02	0.07±0.0.1	0.04±0.02

环境因子Environmental factors	变量Variables	分组Group
环境因子Environmental factors	变量Variables	DB	DN	HB	HN
理化性质 Soil physical-chemical property	总氮TN/%	1 805.37±293.06	1 814.36±152.19	1 676.88±248.44	1 777.86±137.70
	铵态氮NH₄^-N /（mg·kg^-1）	4.41±1.11	4.22±0.54	4.52±0.94	4.30±1.00
	硝态氮NO₃^-N /（mg·kg^-1）	38.87±2.85	39.97±1.70	41.84±4.35	40.06±2.08
	有机碳TOC/%	2.29±0.30	2.24±0.29	2.23±0.31	2.17±0.28
土壤性质 Soil property	温度MAT/℃	13.49±3.54	11.88±0.18	13.98±4.36	11.87±0.20
	水分SMC/%	9.81±1.06	10.63±1.31	10.63±1.23	10.70±1.45
	电导率EC/（us·cm^-1）	0.06±0.04	0.04±0.02	0.07±0.0.1	0.04±0.02

整体比较Global test			两两比较Pairwise composition
整体比较Global test			B	N	HB	HN	DB	DN
MRPP	0.415 8	B		0.6	0.048	0.011	0.006	0.025
δ（P-value）	0.001	N	0.291 6		0.004	0.002	0.004	0.008
		HB	0.462 1	0.461 0		0.75	0.504	0.946
		HN	0.359 8	0.317 9	0.513 3		0.049	0.232
		DB	0.362 1	0.320 3	0.516 1	0.392 8		0.368
		DN	0.430 4	0.392 0	0.582 7	0.469 4	0.471 7
ANOSIM	0.423 6	B		0.73	0.011	0.012	0.003	0.03
R（P-value）	0.001	N	-0.055		0.008	0.002	0.001	0.009
		HB	0.488 0	0.678 5		0.284	0.185	0.606
		HN	0.586 6	0.853 3	0.068 7		0.048	0.373
		DB	0.936	0.997 3	0.131 2	0.384		0.48
		DN	0.340 7	0.485 1	-0.06 7	0.018 6	-0.026
PERMANOVA	4.100 5	B		0.504	0.049	0.009	0.011	0.023
F（P-value）	0.001	N	0.962 6		0.016	0.001	0.002	0.008
		HB	3.041 3	4.806 7		0.861	0.424	0.909
		HN	6.581 5	11.153 1	0.541 9		0.045	0.286
		DB	12.739 4	19.585 4	1.021 9	2.477 6		0.316
		DN	4.704 1	7.176 4	0.416 5	1.189 4	1.150 2