生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 204-215.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1066
颜珲璘1(), 芦光新1(), 邓晔2, 顾松松2, 颜程良2, 马坤1, 赵阳安1, 张海娟1, 王英成1, 周学丽3, 窦声云3
收稿日期:
2021-08-19
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
颜珲璘,女,博士研究生,研究方向:草地生态与环境保护;E-mail:基金资助:
YAN Hui-lin1(), LU Guang-xin1(), DENG Ye2, GU Song-song2, YAN Cheng-liang2, MA Kun1, ZHAO Yang-an1, ZHANG Hai-juan1, WANG Ying-cheng1, ZHOU Xue-li3, DOU Sheng-yun3
Received:
2021-08-19
Published:
2022-10-26
Online:
2022-11-11
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。
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.
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 |
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 |
苜蓿‘北林201’Medicago sativa cv.‘Beilin201’ | 3 | D2 |
表1 试验设计
Table 1 Experimental design
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 |
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 |
苜蓿‘北林201’Medicago sativa cv.‘Beilin201’ | 3 | D2 |
环境因子Environmental factors | 变量Variables | 分组Group | ||||
---|---|---|---|---|---|---|
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 |
表2 土壤理化性质
Table 2 Soil physical and chemical properties
环境因子Environmental factors | 变量Variables | 分组Group | ||||
---|---|---|---|---|---|---|
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 |
图2 土壤原核生物微生物群落稀释曲线和共有和独有OTUs 的韦恩 a:测序深度已基本满足下游分析;b:不同分组共有和独有OTU 可视化。B:根周拌种;N:根周不拌种。下同
Fig. 2 Rarefaction curves for soil prokaryotic microbial communities and Venn diagram showing the unique and shared OTUs a: Sequencing depth has basically satisfied downstream analysis. b: Visualization of common and unique OTUs in different groups. B: Bulk soil seed dressing. N: Bulk soil without seed dressing. The same below
图3 α多样性分析 a:Shannon指数分析;b:Observed_richness指数分析;c:Inverse Simpson指数分析;d:Chao值分析。*表示差异显著,其中*P<0.05,**P<0.01,***P<0.001。下同
Fig. 3 α diversity analysis a:Shannon index analysis. b:Observed_richness index analysis. c:Inverse Simpson index analysis. d:Chao index analysis. * indicates significant differences,* P<0.05,** P<0.01,and *** P<0.001. The same below
图5 根周与根际原核微生物群落结构非度量多维尺度分析(NMDS) a:基于Bary-Curtis距离的禾本科NMDS;b:基于Bary-Curtis距离的豆科NMDS
Fig. 5 NMDS of bulk and rhizosphere soil prokaryotic community structure a:Gramineous NMDS based on Bary-Curtis distance. b:Legume NMDS based on Bary-Curtis distance
整体比较Global test | 两两比较Pairwise composition | |||||||
---|---|---|---|---|---|---|---|---|
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 |
表3 基于原核微生物群落Bary-Curtis距离的不相似检验
Table 3 Dissimilarity test based on Bary-Curtis distance of soil prokaryotic communities
整体比较Global test | 两两比较Pairwise composition | |||||||
---|---|---|---|---|---|---|---|---|
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 |
高水平表型 High-level phenotypes | 根周土 Bulk soil | 根际土 Rhizosphere soil | |||||
---|---|---|---|---|---|---|---|
B | N | HB | HN | DB | DN | ||
需氧Aerobic | 99 | 117 | 51 | 19 | 13 | 36 | |
厌氧Anaerobic | 48 | 50 | 30 | 24 | 13 | 30 | |
兼性厌氧 Facultatively_anaerobic | 25 | 18 | 14 | 9 | 3 | 10 | |
革兰氏阳性Gram positive | 39 | 48 | 116 | 84 | 39 | 102 | |
革兰氏阴性Gram_negative | 233 | 22 | 21 | 15 | 6 | 14 |
表4 微生物表型预测结果
Table 4 Bugbase results
高水平表型 High-level phenotypes | 根周土 Bulk soil | 根际土 Rhizosphere soil | |||||
---|---|---|---|---|---|---|---|
B | N | HB | HN | DB | DN | ||
需氧Aerobic | 99 | 117 | 51 | 19 | 13 | 36 | |
厌氧Anaerobic | 48 | 50 | 30 | 24 | 13 | 30 | |
兼性厌氧 Facultatively_anaerobic | 25 | 18 | 14 | 9 | 3 | 10 | |
革兰氏阳性Gram positive | 39 | 48 | 116 | 84 | 39 | 102 | |
革兰氏阴性Gram_negative | 233 | 22 | 21 | 15 | 6 | 14 |
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