磷差异性调控水稻根际nirK/nirS型反硝化菌组成与丰度
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摘要
磷作为一种重要的生命元素,对反硝化微生物的活性和功能有重要影响.反硝化功能基因nir K和nir S是编码亚硝酸还原酶的两种同工酶基因,但是,磷对具有同种功能的nir K和nir S型反硝化菌的调控是否存在差异尚不十分清楚.本文采集严重缺磷红壤性水稻土设置水稻盆栽试验,通过外源添加磷肥设置对照(CK,P:0 mg·kg-1),低磷(P1,P:15 mg·kg-1),高磷(P2,P:30 mg·kg-1)这3个磷添加水平,研究不同磷水平对水稻亚硝酸还原酶基因的组成与丰度的调控作用.结果表明,在长期缺磷土壤上施用磷肥对含亚硝酸还原酶基因nir K和nir S的细菌种群的调控作用有明显差异.不管是根际还是非根际土,含nir S的微生物种群均对施磷水平表现敏感,尤其是高磷(P2)水平,施磷可导致nir S丰度提高2～3倍,同时显著改变nir S型反硝化微生物的组成结构.相比之下,施磷后含nir K基因的微生物组成结构和丰度变异较小.与非根际土壤相比,高磷水平条件下根际土中nir S的基因拷贝数和群落结构均发生了显著变化,缺磷和低磷条件下水稻生长只引起根际土nir S种群组成结构发生显著变化,但其丰度与非根际无显著差异.但不同磷水平条件下nir K的基因丰度和组成结构在根际和非根际土之间几乎无显著变化.综上所述,在严重缺磷水稻土中加施磷肥会显著提高水稻根际和非根际土中nir K和nir S型反硝化菌数量,并改变其种群组成结构,且nir S比nir K型种群响应更敏感.不同磷水平条件下的水稻根系生长均显著改变了根际土壤中nir K和nir S种群组成结构,但除了在高磷水平条件下显著增加了nir S丰度外,对nir K和nir S丰度均影响较小.研究结果可为进一步深入探究施肥对土壤反硝化过程的影响提供理论依据.
Phosphorus is an essential life element,which can affect the activities and functions of denitrifiers. Both nir K and nir S genes can code nitrite reductase; however,it remains unclear whether nir K-and nir S-containing denitrifers respond differentially to changes in the availability of phosphorus in paddy soil. In this study,P-deficient paddy soil was used to grow rice plants. Three phosphorus levels established by applying P fertilizer at a rate of 0 mg·kg-1( CK),15 mg·kg-1( P1),and 30 mg·kg-1( P2),respectively. The abundance and community structure of nir K-and nir S-containing denitrifers were determined using quantitative PCR and highthroughput sequencing techniques. Results indicated that nir K-and nir S-containing communities responded differentially to changes in the P levels. The nir S-containing communities are more sensitive to the changes in P in both rhizosphere and bulk soil samples. In addition,the abundance of nir S genes was 2-3 times higher in the P2 treatment than in the CK treatment. Furthermore,the nir S community structure is also clearly differed from the CK treatment. However,P addition only induced partial modification of the community structure and abundance of nir K-containing denitrifiers. Moreover,compared to the bulk soil with each phosphorus level,the nir S community structure in the rhizosphere soil changed significantly; however,only the P2 treatment induced significant increases in the abundance of the nir S gene. In contrast,no significant differences in the abundance and composition of nir K-containing denitrifers were detected between rhizosphere and bulk soils under different phosphorus levels. Collectively,application of phosphate fertilizer in P-deficient paddy soil could significantly increase the abundance of nir K-and nir S-containing denitrifiers,changing their community structures, with nir S-type showing a greater sensitivity than nir K-type denitrifiers. In comparison, the denitrifying communities in the rhizosphere were more sensitive to variable P levels than that in the bulk soil. Compared to bulk soils,rice growth shifted the community structure of nir S-and nir K-containing denitrifiers in rhizosphere soils at each level of P,but failed to induce significant changes in their abundance( except for P2) that could cause a significant increase in nir S abundance. These results could provide a theoretical basis for exploring the effects of fertilization on soil denitrification.
Phosphorus is an essential life element,which can affect the activities and functions of denitrifiers. Both nir K and nir S genes can code nitrite reductase; however,it remains unclear whether nir K-and nir S-containing denitrifers respond differentially to changes in the availability of phosphorus in paddy soil. In this study,P-deficient paddy soil was used to grow rice plants. Three phosphorus levels established by applying P fertilizer at a rate of 0 mg·kg-1( CK),15 mg·kg-1( P1),and 30 mg·kg-1( P2),respectively. The abundance and community structure of nir K-and nir S-containing denitrifers were determined using quantitative PCR and highthroughput sequencing techniques. Results indicated that nir K-and nir S-containing communities responded differentially to changes in the P levels. The nir S-containing communities are more sensitive to the changes in P in both rhizosphere and bulk soil samples. In addition,the abundance of nir S genes was 2-3 times higher in the P2 treatment than in the CK treatment. Furthermore,the nir S community structure is also clearly differed from the CK treatment. However,P addition only induced partial modification of the community structure and abundance of nir K-containing denitrifiers. Moreover,compared to the bulk soil with each phosphorus level,the nir S community structure in the rhizosphere soil changed significantly; however,only the P2 treatment induced significant increases in the abundance of the nir S gene. In contrast,no significant differences in the abundance and composition of nir K-containing denitrifers were detected between rhizosphere and bulk soils under different phosphorus levels. Collectively,application of phosphate fertilizer in P-deficient paddy soil could significantly increase the abundance of nir K-and nir S-containing denitrifiers,changing their community structures, with nir S-type showing a greater sensitivity than nir K-type denitrifiers. In comparison, the denitrifying communities in the rhizosphere were more sensitive to variable P levels than that in the bulk soil. Compared to bulk soils,rice growth shifted the community structure of nir S-and nir K-containing denitrifiers in rhizosphere soils at each level of P,but failed to induce significant changes in their abundance( except for P2) that could cause a significant increase in nir S abundance. These results could provide a theoretical basis for exploring the effects of fertilization on soil denitrification.
引文
[1] Rausch C, Bucher M. Molecular mechanisms of phosphate transport in plants[J]. Planta,2002,216(1):23-37.
[2] Holford I C R. Soil phosphorus:its measurement,and its uptake by plants[J]. Australian Journal of Soil Research,1997,35(2):227-240.
[3]陆雅海,张福锁.根际微生物研究进展[J].土壤,2006,38(2):113-121.Lu Y H,Zhang F S. The advances in rhizosphere microbiology[J]. Soils,2006,38(2):113-121.
[4] Philippot L,Hallin S,Brjesson G,et al. Biochemical cycling in the rhizosphere having an impact on global change[J]. Plant and Soil,2009,321(1-2):61-81.
[5] Dotaniya M L,Meena V D. Rhizosphere effect on nutrient availability in soil and its uptake by plants:a review[J].Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2015,85(1):1-12.
[6]贺纪正,张丽梅.土壤氮素转化的关键微生物过程及机制[J].微生物学通报,2013,40(1):98-108.He J Z,Zhang L M. Key processes and microbial mechanisms of soil nitrogen transformation[J]. Microbiology China,2013,40(1):98-108.
[7] Zhou S L,Huang T L,Zhang C H,et al. Illumina Mi Seq sequencing reveals the community composition of nirS-type and nirK-type denitrifiers in Zhoucun reservoir-a large shallow eutrophic reservoir in northern China[J]. RSC Advances,2016,6(94):91517-91528.
[8] Zumft W G. Cell biology and molecular basis of denitrification[J]. Microbiology and Molecular Biology Reviews,1997,61(4):533-616.
[9]郭丽芸,时飞,杨柳燕.反硝化菌功能基因及其分子生态学研究进展[J].微生物学通报,2011,38(4):583-590.Guo L Y,Shi F,Yang L Y. Advances in functional genes and molecular ecology in denitrifiers[J]. Microbiology China,2011,38(4):583-590.
[10] Heylen K,Gevers D,Vanparys B,et al. The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers[J]. Environmental Microbiology,2006,8(11):2012-2021.
[11] Bárta J,MelichováT,Vaněk D,et al. Effect of pH and dissolved organic matter on the abundance of nirK and nirS denitrifiers in spruce forest soil[J]. Biogeochemistry,2010,101(1-3):123-132.
[12] Chen Z,Luo X Q,Hu R G,et al. Impact of long-term fertilization on the composition of denitrifier communities based on nitrite reductase analyses in a paddy soil[J]. Microbial Ecology,2010,60(4):850-861.
[13] Chen Z,Liu J B,Wu M N,et al. Differentiated response of denitrifying communities to fertilization regime in paddy soil[J].Microbial Ecology,2012,63(2):446-459.
[14] Liu J B,Hou H J,Sheng R,et al. Denitrifying communities differentially respond to flooding drying cycles in paddy soils[J].Applied Soil Ecology,2012,62:155-162.
[15]尹昌,范分良,李兆君,等.长期施用有机和无机肥对黑土nirS型反硝化菌种群结构和丰度的影响[J].环境科学,2012,33(11):3967-3975.Yin C, Fan F L, Li Z J, et al. Influences of long-term application of organic and inorganic fertilizers on the composition and abundance of nirS-type denitrifiers in black soil[J].Environmental Science,2012,33(11):3967-3975.
[16] Sheng R,Meng D L,Wu M N,et al. Effect of agricultural land use change on community composition of bacteria and ammonia oxidizers[J]. Journal of Soils and Sediments,2013,13(7):1246-1256.
[17] He M Z,Dijkstra F A. Phosphorus addition enhances loss of nitrogen in a phosphorus-poor soil[J]. Soil Biology and Biochemistry,2015,82:99-106.
[18] Mori T,Ohta S,Ishizuka S,et al. Phosphorus application reduces N2O emissions from tropical leguminous plantation soil when phosphorus uptake is occurring[J]. Biology and Fertility of Soils,2014,50(1):45-51.
[19] Duan Y H,Shi X J,Li S L,et al. Nitrogen use efficiency as affected by phosphorus and potassium in long-term rice and wheat experiments[J]. Journal of Integrative Agriculture,2014,13(3):588-596.
[20]鲍士旦.土壤农化分析[M].(第三版).北京:中国农业出版社,2000. 49-56,79-83.
[21] Henry S,Baudoin E,López-Gutiérrez J C,et al. Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR[J]. Journal of Microbiological Methods,2004,59(3):327-335.
[22] Hallin S,Lindgren P E. PCR detection of genes encoding nitrite reductase in denitrifying bacteria[J]. Applied and Environmental Microbiology,1999,65(4):1652-1657.
[23] Michotey V,Méjean V,Bonin P. Comparison of methods for quantification of cytochrome cd1-denitrifying bacteria in environmental marine samples[J]. Applied and Environmental Microbiology,2000,66(4):1564-1571.
[24] Throbck I N,Enwall K,Jarvis,et al. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J]. FEMS Microbiology Ecology,2004,49(3):401-417.
[25] Palmer K, Biasi C, Horn M A. Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra[J]. The ISME Journal,2012,6(5):1058-1077.
[26] Cleveland C C,Townsend A R. Nutrient additions to a tropical rain forest drive substantial soil carbon dioxide losses to the atmosphere[J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(27):10316-10321.
[27] Wei X M,Hu Y J,Peng P Q,et al. Effect of P stoichiometry on the abundance of nitrogen-cycle genes in phosphorus-limited paddy soil[J]. Biology and Fertility of Soils,2017,53(7):767-776.
[28] Jones C M, Hallin S. Ecological and evolutionary factors underlying global and local assembly of denitrifier communities[J]. The ISME Journal,2010,4(5):633-641.
[29] Tang Y Q,Zhang X Y,Li D D,et al. Impacts of nitrogen and phosphorus additions on the abundance and community structure of ammonia oxidizers and denitrifying bacteria in Chinese fir plantations[J]. Soil Biology and Biochemistry,2016,103:284-293.
[30] Brenzinger K,Drsch P,Braker G. pH-driven shifts in overall and transcriptionally active denitrifiers control gaseous product stoichiometry in growth experiments with extracted bacteria from soil[J]. Frontiers in Microbiology,2015,6:961.
[31] Herold M B,Giles M E,Alexander C J,et al. Variable response of nirK and nirS containing denitrifier communities to long-term pH manipulation and cultivation[J]. FEMS Microbiology Letters,2018,365(7),doi:10. 1093/femsle/fny035.
[32] Sarilmiser H K,Ates O,Ozdemir G,et al. Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T[J]. Journal of Bioscience and Bioengineering,2015,119(4):455-463.
[33] Allers E,Gómez‐Consarnau L,Pinhassi J,et al. Response of Alteromonadaceae and Rhodobacteriaceae to glucose and phosphorus manipulation in marine mesocosms[J].Environmental Microbiology,2007,9(10):2417-2429.
[34] Sánchez O,Koblíek M,Gasol J M,et al. Effects of grazing,phosphorus and light on the growth rates of major bacterioplankton taxa in the coastal NW Mediterranean[J]. Environmental Microbiology Reports,2017,9(3):300-309.
[35] Yin C,Fan F L,Song A L,et al. Different denitrification potential of aquic brown soil in northeast China under inorganic and organic fertilization accompanied by distinct changes of nirSand nirK-denitrifying bacterial community[J]. European Journal of Soil Biology,2014,65:47-56.
[36] Wu H L,Wang X Z,He X J,et al. Effects of root exudates on denitrifier gene abundance,community structure and activity in a micro-polluted constructed wetland[J]. Science of the Total Environment,2017,598:697-703.
[37]罗永清,赵学勇,李美霞.植物根系分泌物生态效应及其影响因素研究综述[J].应用生态学报,2012,23(12):3496-3504.Luo Y Q,Zhao X Y,Li M X. Ecological effect of plant root exudates and related affecting factors:a review[J]. Chinese Journal of Applied Ecology,2012,23(12):3496-3504.
[38] Coyne M S,Arunakumari A,Averill B A,et al. Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria[J].Applied and Environmental Microbiology,1989,55(11):2924-2931.
[39]朱丽霞,章家恩,刘文高.根系分泌物与根际微生物相互作用研究综述[J].生态环境,2003,12(1):102-105.Zhu L X,Zhang J E,Liu W G. Review of studies on interactions between root exudates and rhizopheric microorganisms[J].Ecology and Environment,2003,12(1):102-105.
[40] Rovira D A. Interactions between plant roots and soil microorganisms[J]. Annual Review of Microbiology,1965,19:241-266.
[41] Phillips L A,Armstrong S A,Headley J V,et al. Shifts in rootassociated microbial communities of Typha latifolia growing in naphthenic acids and relationship to plant health[J].International Journal of Phytoremediation,2010,12(8):745-760.
[2] Holford I C R. Soil phosphorus:its measurement,and its uptake by plants[J]. Australian Journal of Soil Research,1997,35(2):227-240.
[3]陆雅海,张福锁.根际微生物研究进展[J].土壤,2006,38(2):113-121.Lu Y H,Zhang F S. The advances in rhizosphere microbiology[J]. Soils,2006,38(2):113-121.
[4] Philippot L,Hallin S,Brjesson G,et al. Biochemical cycling in the rhizosphere having an impact on global change[J]. Plant and Soil,2009,321(1-2):61-81.
[5] Dotaniya M L,Meena V D. Rhizosphere effect on nutrient availability in soil and its uptake by plants:a review[J].Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2015,85(1):1-12.
[6]贺纪正,张丽梅.土壤氮素转化的关键微生物过程及机制[J].微生物学通报,2013,40(1):98-108.He J Z,Zhang L M. Key processes and microbial mechanisms of soil nitrogen transformation[J]. Microbiology China,2013,40(1):98-108.
[7] Zhou S L,Huang T L,Zhang C H,et al. Illumina Mi Seq sequencing reveals the community composition of nirS-type and nirK-type denitrifiers in Zhoucun reservoir-a large shallow eutrophic reservoir in northern China[J]. RSC Advances,2016,6(94):91517-91528.
[8] Zumft W G. Cell biology and molecular basis of denitrification[J]. Microbiology and Molecular Biology Reviews,1997,61(4):533-616.
[9]郭丽芸,时飞,杨柳燕.反硝化菌功能基因及其分子生态学研究进展[J].微生物学通报,2011,38(4):583-590.Guo L Y,Shi F,Yang L Y. Advances in functional genes and molecular ecology in denitrifiers[J]. Microbiology China,2011,38(4):583-590.
[10] Heylen K,Gevers D,Vanparys B,et al. The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers[J]. Environmental Microbiology,2006,8(11):2012-2021.
[11] Bárta J,MelichováT,Vaněk D,et al. Effect of pH and dissolved organic matter on the abundance of nirK and nirS denitrifiers in spruce forest soil[J]. Biogeochemistry,2010,101(1-3):123-132.
[12] Chen Z,Luo X Q,Hu R G,et al. Impact of long-term fertilization on the composition of denitrifier communities based on nitrite reductase analyses in a paddy soil[J]. Microbial Ecology,2010,60(4):850-861.
[13] Chen Z,Liu J B,Wu M N,et al. Differentiated response of denitrifying communities to fertilization regime in paddy soil[J].Microbial Ecology,2012,63(2):446-459.
[14] Liu J B,Hou H J,Sheng R,et al. Denitrifying communities differentially respond to flooding drying cycles in paddy soils[J].Applied Soil Ecology,2012,62:155-162.
[15]尹昌,范分良,李兆君,等.长期施用有机和无机肥对黑土nirS型反硝化菌种群结构和丰度的影响[J].环境科学,2012,33(11):3967-3975.Yin C, Fan F L, Li Z J, et al. Influences of long-term application of organic and inorganic fertilizers on the composition and abundance of nirS-type denitrifiers in black soil[J].Environmental Science,2012,33(11):3967-3975.
[16] Sheng R,Meng D L,Wu M N,et al. Effect of agricultural land use change on community composition of bacteria and ammonia oxidizers[J]. Journal of Soils and Sediments,2013,13(7):1246-1256.
[17] He M Z,Dijkstra F A. Phosphorus addition enhances loss of nitrogen in a phosphorus-poor soil[J]. Soil Biology and Biochemistry,2015,82:99-106.
[18] Mori T,Ohta S,Ishizuka S,et al. Phosphorus application reduces N2O emissions from tropical leguminous plantation soil when phosphorus uptake is occurring[J]. Biology and Fertility of Soils,2014,50(1):45-51.
[19] Duan Y H,Shi X J,Li S L,et al. Nitrogen use efficiency as affected by phosphorus and potassium in long-term rice and wheat experiments[J]. Journal of Integrative Agriculture,2014,13(3):588-596.
[20]鲍士旦.土壤农化分析[M].(第三版).北京:中国农业出版社,2000. 49-56,79-83.
[21] Henry S,Baudoin E,López-Gutiérrez J C,et al. Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR[J]. Journal of Microbiological Methods,2004,59(3):327-335.
[22] Hallin S,Lindgren P E. PCR detection of genes encoding nitrite reductase in denitrifying bacteria[J]. Applied and Environmental Microbiology,1999,65(4):1652-1657.
[23] Michotey V,Méjean V,Bonin P. Comparison of methods for quantification of cytochrome cd1-denitrifying bacteria in environmental marine samples[J]. Applied and Environmental Microbiology,2000,66(4):1564-1571.
[24] Throbck I N,Enwall K,Jarvis,et al. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J]. FEMS Microbiology Ecology,2004,49(3):401-417.
[25] Palmer K, Biasi C, Horn M A. Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra[J]. The ISME Journal,2012,6(5):1058-1077.
[26] Cleveland C C,Townsend A R. Nutrient additions to a tropical rain forest drive substantial soil carbon dioxide losses to the atmosphere[J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(27):10316-10321.
[27] Wei X M,Hu Y J,Peng P Q,et al. Effect of P stoichiometry on the abundance of nitrogen-cycle genes in phosphorus-limited paddy soil[J]. Biology and Fertility of Soils,2017,53(7):767-776.
[28] Jones C M, Hallin S. Ecological and evolutionary factors underlying global and local assembly of denitrifier communities[J]. The ISME Journal,2010,4(5):633-641.
[29] Tang Y Q,Zhang X Y,Li D D,et al. Impacts of nitrogen and phosphorus additions on the abundance and community structure of ammonia oxidizers and denitrifying bacteria in Chinese fir plantations[J]. Soil Biology and Biochemistry,2016,103:284-293.
[30] Brenzinger K,Drsch P,Braker G. pH-driven shifts in overall and transcriptionally active denitrifiers control gaseous product stoichiometry in growth experiments with extracted bacteria from soil[J]. Frontiers in Microbiology,2015,6:961.
[31] Herold M B,Giles M E,Alexander C J,et al. Variable response of nirK and nirS containing denitrifier communities to long-term pH manipulation and cultivation[J]. FEMS Microbiology Letters,2018,365(7),doi:10. 1093/femsle/fny035.
[32] Sarilmiser H K,Ates O,Ozdemir G,et al. Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T[J]. Journal of Bioscience and Bioengineering,2015,119(4):455-463.
[33] Allers E,Gómez‐Consarnau L,Pinhassi J,et al. Response of Alteromonadaceae and Rhodobacteriaceae to glucose and phosphorus manipulation in marine mesocosms[J].Environmental Microbiology,2007,9(10):2417-2429.
[34] Sánchez O,Koblíek M,Gasol J M,et al. Effects of grazing,phosphorus and light on the growth rates of major bacterioplankton taxa in the coastal NW Mediterranean[J]. Environmental Microbiology Reports,2017,9(3):300-309.
[35] Yin C,Fan F L,Song A L,et al. Different denitrification potential of aquic brown soil in northeast China under inorganic and organic fertilization accompanied by distinct changes of nirSand nirK-denitrifying bacterial community[J]. European Journal of Soil Biology,2014,65:47-56.
[36] Wu H L,Wang X Z,He X J,et al. Effects of root exudates on denitrifier gene abundance,community structure and activity in a micro-polluted constructed wetland[J]. Science of the Total Environment,2017,598:697-703.
[37]罗永清,赵学勇,李美霞.植物根系分泌物生态效应及其影响因素研究综述[J].应用生态学报,2012,23(12):3496-3504.Luo Y Q,Zhao X Y,Li M X. Ecological effect of plant root exudates and related affecting factors:a review[J]. Chinese Journal of Applied Ecology,2012,23(12):3496-3504.
[38] Coyne M S,Arunakumari A,Averill B A,et al. Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria[J].Applied and Environmental Microbiology,1989,55(11):2924-2931.
[39]朱丽霞,章家恩,刘文高.根系分泌物与根际微生物相互作用研究综述[J].生态环境,2003,12(1):102-105.Zhu L X,Zhang J E,Liu W G. Review of studies on interactions between root exudates and rhizopheric microorganisms[J].Ecology and Environment,2003,12(1):102-105.
[40] Rovira D A. Interactions between plant roots and soil microorganisms[J]. Annual Review of Microbiology,1965,19:241-266.
[41] Phillips L A,Armstrong S A,Headley J V,et al. Shifts in rootassociated microbial communities of Typha latifolia growing in naphthenic acids and relationship to plant health[J].International Journal of Phytoremediation,2010,12(8):745-760.