The diazotrophic community in oat rhizosphere: effects of legume intercropping and crop growth stage
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摘要
In this study, the abundance, diversity and structure of the diazotrophic community in oat rhizosphere soil in three cropping systems and at two oat growth stages were investigated using real-time PCR and Illumina MiSeq sequencing. The nifH gene abundance in oat-soybean intercropping(OSO) and oat-mungbean intercropping(OMO) was significantly greater than that in sole oat(O), but the nifH gene abundance significantly decreased at the later stage in all the treatments. Alpha diversity indices in OSO and OMO were higher at the heading stage, but lower at the maturity stage than that in O. Bradyrhizobium and Skermanella were the dominant genera identified in all samples, with an average proportion of 35.8% and 12.4%,respectively. The proportion of dominant genera showed significant differences and varied with cropping system and growth stage. Principal component analysis showed that growth stage had a stronger effect than intercropping on the diazotrophic community structure. However,Mantel test and redundancy analysis showed there was no environmental factor significantly correlated to the diazotrophic community structure. Our results demonstrate that intercropping had a weaker effect than growth stage on the abundance, diversity and structure of the diazotrophic community in oat rhizosphere soil.
In this study, the abundance, diversity and structure of the diazotrophic community in oat rhizosphere soil in three cropping systems and at two oat growth stages were investigated using real-time PCR and Illumina MiSeq sequencing. The nifH gene abundance in oat-soybean intercropping(OSO) and oat-mungbean intercropping(OMO) was significantly greater than that in sole oat(O), but the nifH gene abundance significantly decreased at the later stage in all the treatments. Alpha diversity indices in OSO and OMO were higher at the heading stage, but lower at the maturity stage than that in O. Bradyrhizobium and Skermanella were the dominant genera identified in all samples, with an average proportion of 35.8% and 12.4%,respectively. The proportion of dominant genera showed significant differences and varied with cropping system and growth stage. Principal component analysis showed that growth stage had a stronger effect than intercropping on the diazotrophic community structure. However,Mantel test and redundancy analysis showed there was no environmental factor significantly correlated to the diazotrophic community structure. Our results demonstrate that intercropping had a weaker effect than growth stage on the abundance, diversity and structure of the diazotrophic community in oat rhizosphere soil.
In this study, the abundance, diversity and structure of the diazotrophic community in oat rhizosphere soil in three cropping systems and at two oat growth stages were investigated using real-time PCR and Illumina MiSeq sequencing. The nifH gene abundance in oat-soybean intercropping(OSO) and oat-mungbean intercropping(OMO) was significantly greater than that in sole oat(O), but the nifH gene abundance significantly decreased at the later stage in all the treatments. Alpha diversity indices in OSO and OMO were higher at the heading stage, but lower at the maturity stage than that in O. Bradyrhizobium and Skermanella were the dominant genera identified in all samples, with an average proportion of 35.8% and 12.4%,respectively. The proportion of dominant genera showed significant differences and varied with cropping system and growth stage. Principal component analysis showed that growth stage had a stronger effect than intercropping on the diazotrophic community structure. However,Mantel test and redundancy analysis showed there was no environmental factor significantly correlated to the diazotrophic community structure. Our results demonstrate that intercropping had a weaker effect than growth stage on the abundance, diversity and structure of the diazotrophic community in oat rhizosphere soil.
引文
1.Lithourgidis A S,Dordas C A,Damalas C A,Vlachostergios D N.Annual intercrops:an alternative pathway for sustainable agriculture.Australian Journal of Crop Science,2011,5(4):396-410
2.Neugschwandtner G W,Kaul H P.Nitrogen uptake,use and utilization efficiency by oat-pea intercrops.Field Crops Research,2015,179:113-119
3.Gao Y,Duan A W,Sun J S,Li F S,Liu Z G,Liu H,Liu Z D.Crop coefficient and water-use efficiency of winter wheat/spring maize strip intercropping.Field Crops Research,2009,111(1-2):65-73
4.Hauggaard-Nielsen H,Gooding M,Ambus P,Corre-Hellou G,Crozat Y,Dahlmann C,Dibet A,von Fragstein P,Pristeri A,Monti M,Jensen E S.Pea-barley intercropping for efficient symbiotic N2-fixation,soil N acquisition and use of other nutrients in European organic cropping systems.Field Crops Research,2009,113(1):64-71
5.Li L,Tilman D,Lambers H,Zhang F S.Plant diversity and overyielding:insights from belowground facilitation of intercropping in agriculture.New Phytologist,2014,203(1-2):63-69
6.Zhang F S,Li L.Using competitive and facilitative interactions in intercropping systems enhances crop productivity and nutrient-use efficiency.Plant and Soil,2003,248(1):305-312
7.Kennedy I R,Choudhury A T M A,Kecskés M L.Non-symbiotic bacterial diazotrophs in crop-farming systems:can their potential for plant growth promotion be better explained?Soil Biology&Biochemistry,2004,36(8):1229-1244
8.Vitousek P M,Aber J D,Howarth R W,Likens G E,Matson P A,Schindler D W,Schlesinger W H,Tilman D G.Human alteration of the global nitrogen cycle:sources and consequences.Ecological Applications,1997,7(3):737-750
9.Gruber N,Galloway J N.An Earth-system perspective of the global nitrogen cycle.Nature,2008,451(7176):293-296
10.Stacey G,Burris R H,Evans H J.Biological Nitrogen Fixation.New York:Chapman and Hall,1992
11.Raymond J,Siefert J L,Staples C R,Blankenship R E.The natural history of nitrogen fixation.Molecular Biology and Evolution,2004,21(3):541-554
12.Tourova T P,Slobodova N V,Bumazhkin B K,Sukhacheva M V,Sorokin D Y.Diversity of diazotrophs in the sediments of saline and soda lakes analyzed with the use of the nifH gene as a molecular marker.Microbiology,2014,83(5):634-647
13.Berthrong S T,Yeager C M,Gallegos-Graves L,Steven B,Eichorst S A,Jackson R B,Kuske C R.Nitrogen fertilization has a stronger effect on soil nitrogen-fixing bacterial communities than elevated atmospheric CO2.Applied and Environmental Microbiology,2014,80(10):3103-3112
14.Hayden H L,Drake J,Imhof M,Oxley A,Norng S,Mele P M.The abundance of nitrogen cycle genes amoA and nifH depends on landuses and soil types in South-Eastern Australia.Soil Biology&Biochemistry,2010,42(10):1774-1783
15.Li X P,Mu Y H,Cheng Y B,Liu X G,Nian H.Effects of intercropping sugarcane and soybean on growth,rhizosphere soil microbes,nitrogen and phosphorus availability.Acta Physiologiae Plantarum,2013,35(4):1113-1119
16.Yang Z,Yang W,Li S,Hao J,Su Z,Sun M,Gao Z Q,Zhang C L.Variation of bacterial community diversity in rhizosphere soil of sole-cropped versus intercropped heat field after harvest.PLoS One,2016,11(3):1-18
17.Xiao C H,Tang H,Pu L J,Sun D M,Ma J Z,Yu M,Duan R S.Diversity of nitrogenase(nifH)genes pool in soybean field soil after continuous and rotational cropping.Journal of Basic Microbiology,2010,50(4):373-379
18.Pereira e Silva M C,Schloter-Hai B,Schloter M,van Elsas J D,Salles J F.Temporal dynamics of abundance and composition of nitrogen-fixing communities across agricultural soils.PLoS One,2013,8(9):e74500
19.Bao S D.Soil agro-chemistrical analysis.Beijing:China Agricultural Press,2000(in Chinese)
20.Poly F,Monrozier L J,Bally R.Improvement in the RFLPprocedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil.Microbiological Research,2001,152(1):95-103
21.R?sch C,Mergel A,Bothe H.Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil.Applied and Environmental Microbiology,2002,68(8):3818-3829
22.Edgar R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads.Nature Methods,2013,10(10):996-998
23.Edgar R C,Haas B J,Clemente J C,Quince C,Knight R.UCHIMEimproves sensitivity and speed of chimera detection.Bioinformatics,2011,27(16):2194-2200
24.Wang Q,Garrity G M,Tiedje J M,Cole J R.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,2007,73(16):5261-5267
25.Schloss P D,Gevers D,Westcott S L.Reducing the effects of PCRamplification and sequencing artifacts on 16S rRNA-based studies.PLoS One,2011,6(12):e27310
26.Oksanen J,Blanchet F G,Friendly M,Kindt R,Legendre P,McGlinn D,Minchin P R,O’Hara R B,Simpson G L,Solymos P,Stevens M H H,Szoecs E,Wagner H.vegan:Community Ecology Package.R package version 2.4-3,2017
27.Kizilova A K,Titova L V,Kravchenko I K,Iutinskaia G A.Evaluation of the diversity of nitrogen-fixing bacteria in soybean rhizosphere by nifH gene analysis.Microbiology,2012,81(5):621-629
28.Li X L,Penttinen P,Gu Y F,Zhang X P.Diversity of nifH gene in rhizosphere and non-rhizosphere soil of tobacco in Panzhihua,China.Annals of Microbiology,2012,62(3):995-1001
29.Tan Z,Hurek T,Reinhold-Hurek B.Effect of N-fertilization,plant genotype and environmental conditions on nifH gene pools in roots of rice.Environmental Microbiology,2003,5(10):1009-1015
30.Knauth S,Hurek T,Brar D,Reinhold-Hurek B.Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice.Environmental Microbiology,2005,7(11):1725-1733
31.Venieraki A,Dimou M,Vezyri E,Kefalogianni I,Argyris N,Liara G,Pergalis P,Chatzipavlidis I,Katinakis P.Characterization of nitrogen-fixing bacteria isolated from field-grown barley,oat,and wheat.Journal of Microbiology,2011,49(4):525-534
32.Pereira e Silva M C,Semenov A V,van Elsas J D,Salles J F.Seasonal variations in the diversity and abundance of diazotrophic communities across soils.FEMS Microbiology Ecology,2011,77(1):57-68
33.Wang J C,Zhang D,Zhang L,Li J,Raza W,Huang Q W,Shen Q R.Temporal variation of diazotrophic community abundance and structure in surface and subsoil under four fertilization regimes during a wheat growing season.Agriculture,Ecosystems&Environment,2016,216:116-124
34.Hai B,Diallo N H,Sall S,Haesler F,Schauss K,Bonzi M,Assigbetse K,Chotte J L,Munch J C,Schloter M.Quantification of key genes steering the microbial nitrogen cycle in the rhizosphere of sorghum cultivars in tropical agroecosystems.Applied and Environmental Microbiology,2009,75(15):4993-5000
35.Soares R A,Roesch L F W,Zanatta G,de Oliveira Camargo F A,Passaglia L M P.Occurrence and distribution of nitrogen fixing bacterial community associated with oat(Avena sativa)assessed by molecular and microbiological techniques.Applied Soil Ecology,2006,33(3):221-234
36.Nelson D R,Mele P M.The impact of crop residue amendments and lime on microbial community structure and nitrogen-fixing bacteria in the wheat rhizosphere.Australian Journal of Soil Research,2006,44(4):319-329
37.de Cássia Pereira e Silva M.The normal operating range of soil functioning:understanding the natural fluctuations of N cycling communities.Collected thesis,the Netherlands:Uiversity of Groningen,2013
38.Zhang N N,Sun Y M,Wang E T,Yang J S,Yuan H L,Scow K M.Effects of intercropping and Rhizobial inoculation on the ammoniaoxidizing microorganisms in rhizospheres of maize and faba bean plants.Applied Soil Ecology,2015,85:76-85
39.Bürgmann H,Meier S,Bunge M,Widmer F,Zeyer J.Effects of model root exudates on structure and activity of a soil diazotroph community.Environmental Microbiology,2005,7(11):1711-1724
40.Rodrigues Coelho M R,de Vos M,Carneiro N P,Marriel I E?,Paiva E,Seldin L.Diversity of nifH gene pools in the rhizosphere of two cultivars of sorghum(Sorghum bicolor)treated with contrasting levels of nitrogen fertilizer.FEMS Microbiology Letters,2008,279(1):15-22
41.Wang S,Gonzalez P G,Ye J,Huang D F.Abundance and diversity of nitrogen-fixing bacteria in rhizosphere and bulk paddy soil under different duration of organic management.World Journal of Microbiology&Biotechnology,2012,28(2):493-503
42.Gutiérrez-Zamora M L,Martínez-Romero E.Natural endophytic association between Rhizobium etli and maize(Zea mays L.).Journal of Biotechnology,2001,91(2-3):117-126
43.Patra A K,Abbadie L,Clays-Josserand A,Degrange V,Grayston SJ,Guillaumaud N,Loiseau P,Louault F,Mahmood S,Nazaret S,Philippot L,Poly F,Prosser J I,Le Roux X.Effects of management regime and plant species on the enzyme activity and genetic structure of N-fixing,denitrifying and nitrifying bacterial communities in grassland soils.Environmental Microbiology,2006,8(6):1005-1016
44.Orr C H,Leifert C,Cummings S P,Cooper J M.Impacts of organic and conventional crop management on diversity and activity of freeliving nitrogen fixing bacteria and total bacteria are subsidiary to temporal effects.PLoS One,2012,7(12):e52891
2.Neugschwandtner G W,Kaul H P.Nitrogen uptake,use and utilization efficiency by oat-pea intercrops.Field Crops Research,2015,179:113-119
3.Gao Y,Duan A W,Sun J S,Li F S,Liu Z G,Liu H,Liu Z D.Crop coefficient and water-use efficiency of winter wheat/spring maize strip intercropping.Field Crops Research,2009,111(1-2):65-73
4.Hauggaard-Nielsen H,Gooding M,Ambus P,Corre-Hellou G,Crozat Y,Dahlmann C,Dibet A,von Fragstein P,Pristeri A,Monti M,Jensen E S.Pea-barley intercropping for efficient symbiotic N2-fixation,soil N acquisition and use of other nutrients in European organic cropping systems.Field Crops Research,2009,113(1):64-71
5.Li L,Tilman D,Lambers H,Zhang F S.Plant diversity and overyielding:insights from belowground facilitation of intercropping in agriculture.New Phytologist,2014,203(1-2):63-69
6.Zhang F S,Li L.Using competitive and facilitative interactions in intercropping systems enhances crop productivity and nutrient-use efficiency.Plant and Soil,2003,248(1):305-312
7.Kennedy I R,Choudhury A T M A,Kecskés M L.Non-symbiotic bacterial diazotrophs in crop-farming systems:can their potential for plant growth promotion be better explained?Soil Biology&Biochemistry,2004,36(8):1229-1244
8.Vitousek P M,Aber J D,Howarth R W,Likens G E,Matson P A,Schindler D W,Schlesinger W H,Tilman D G.Human alteration of the global nitrogen cycle:sources and consequences.Ecological Applications,1997,7(3):737-750
9.Gruber N,Galloway J N.An Earth-system perspective of the global nitrogen cycle.Nature,2008,451(7176):293-296
10.Stacey G,Burris R H,Evans H J.Biological Nitrogen Fixation.New York:Chapman and Hall,1992
11.Raymond J,Siefert J L,Staples C R,Blankenship R E.The natural history of nitrogen fixation.Molecular Biology and Evolution,2004,21(3):541-554
12.Tourova T P,Slobodova N V,Bumazhkin B K,Sukhacheva M V,Sorokin D Y.Diversity of diazotrophs in the sediments of saline and soda lakes analyzed with the use of the nifH gene as a molecular marker.Microbiology,2014,83(5):634-647
13.Berthrong S T,Yeager C M,Gallegos-Graves L,Steven B,Eichorst S A,Jackson R B,Kuske C R.Nitrogen fertilization has a stronger effect on soil nitrogen-fixing bacterial communities than elevated atmospheric CO2.Applied and Environmental Microbiology,2014,80(10):3103-3112
14.Hayden H L,Drake J,Imhof M,Oxley A,Norng S,Mele P M.The abundance of nitrogen cycle genes amoA and nifH depends on landuses and soil types in South-Eastern Australia.Soil Biology&Biochemistry,2010,42(10):1774-1783
15.Li X P,Mu Y H,Cheng Y B,Liu X G,Nian H.Effects of intercropping sugarcane and soybean on growth,rhizosphere soil microbes,nitrogen and phosphorus availability.Acta Physiologiae Plantarum,2013,35(4):1113-1119
16.Yang Z,Yang W,Li S,Hao J,Su Z,Sun M,Gao Z Q,Zhang C L.Variation of bacterial community diversity in rhizosphere soil of sole-cropped versus intercropped heat field after harvest.PLoS One,2016,11(3):1-18
17.Xiao C H,Tang H,Pu L J,Sun D M,Ma J Z,Yu M,Duan R S.Diversity of nitrogenase(nifH)genes pool in soybean field soil after continuous and rotational cropping.Journal of Basic Microbiology,2010,50(4):373-379
18.Pereira e Silva M C,Schloter-Hai B,Schloter M,van Elsas J D,Salles J F.Temporal dynamics of abundance and composition of nitrogen-fixing communities across agricultural soils.PLoS One,2013,8(9):e74500
19.Bao S D.Soil agro-chemistrical analysis.Beijing:China Agricultural Press,2000(in Chinese)
20.Poly F,Monrozier L J,Bally R.Improvement in the RFLPprocedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil.Microbiological Research,2001,152(1):95-103
21.R?sch C,Mergel A,Bothe H.Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil.Applied and Environmental Microbiology,2002,68(8):3818-3829
22.Edgar R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads.Nature Methods,2013,10(10):996-998
23.Edgar R C,Haas B J,Clemente J C,Quince C,Knight R.UCHIMEimproves sensitivity and speed of chimera detection.Bioinformatics,2011,27(16):2194-2200
24.Wang Q,Garrity G M,Tiedje J M,Cole J R.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,2007,73(16):5261-5267
25.Schloss P D,Gevers D,Westcott S L.Reducing the effects of PCRamplification and sequencing artifacts on 16S rRNA-based studies.PLoS One,2011,6(12):e27310
26.Oksanen J,Blanchet F G,Friendly M,Kindt R,Legendre P,McGlinn D,Minchin P R,O’Hara R B,Simpson G L,Solymos P,Stevens M H H,Szoecs E,Wagner H.vegan:Community Ecology Package.R package version 2.4-3,2017
27.Kizilova A K,Titova L V,Kravchenko I K,Iutinskaia G A.Evaluation of the diversity of nitrogen-fixing bacteria in soybean rhizosphere by nifH gene analysis.Microbiology,2012,81(5):621-629
28.Li X L,Penttinen P,Gu Y F,Zhang X P.Diversity of nifH gene in rhizosphere and non-rhizosphere soil of tobacco in Panzhihua,China.Annals of Microbiology,2012,62(3):995-1001
29.Tan Z,Hurek T,Reinhold-Hurek B.Effect of N-fertilization,plant genotype and environmental conditions on nifH gene pools in roots of rice.Environmental Microbiology,2003,5(10):1009-1015
30.Knauth S,Hurek T,Brar D,Reinhold-Hurek B.Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice.Environmental Microbiology,2005,7(11):1725-1733
31.Venieraki A,Dimou M,Vezyri E,Kefalogianni I,Argyris N,Liara G,Pergalis P,Chatzipavlidis I,Katinakis P.Characterization of nitrogen-fixing bacteria isolated from field-grown barley,oat,and wheat.Journal of Microbiology,2011,49(4):525-534
32.Pereira e Silva M C,Semenov A V,van Elsas J D,Salles J F.Seasonal variations in the diversity and abundance of diazotrophic communities across soils.FEMS Microbiology Ecology,2011,77(1):57-68
33.Wang J C,Zhang D,Zhang L,Li J,Raza W,Huang Q W,Shen Q R.Temporal variation of diazotrophic community abundance and structure in surface and subsoil under four fertilization regimes during a wheat growing season.Agriculture,Ecosystems&Environment,2016,216:116-124
34.Hai B,Diallo N H,Sall S,Haesler F,Schauss K,Bonzi M,Assigbetse K,Chotte J L,Munch J C,Schloter M.Quantification of key genes steering the microbial nitrogen cycle in the rhizosphere of sorghum cultivars in tropical agroecosystems.Applied and Environmental Microbiology,2009,75(15):4993-5000
35.Soares R A,Roesch L F W,Zanatta G,de Oliveira Camargo F A,Passaglia L M P.Occurrence and distribution of nitrogen fixing bacterial community associated with oat(Avena sativa)assessed by molecular and microbiological techniques.Applied Soil Ecology,2006,33(3):221-234
36.Nelson D R,Mele P M.The impact of crop residue amendments and lime on microbial community structure and nitrogen-fixing bacteria in the wheat rhizosphere.Australian Journal of Soil Research,2006,44(4):319-329
37.de Cássia Pereira e Silva M.The normal operating range of soil functioning:understanding the natural fluctuations of N cycling communities.Collected thesis,the Netherlands:Uiversity of Groningen,2013
38.Zhang N N,Sun Y M,Wang E T,Yang J S,Yuan H L,Scow K M.Effects of intercropping and Rhizobial inoculation on the ammoniaoxidizing microorganisms in rhizospheres of maize and faba bean plants.Applied Soil Ecology,2015,85:76-85
39.Bürgmann H,Meier S,Bunge M,Widmer F,Zeyer J.Effects of model root exudates on structure and activity of a soil diazotroph community.Environmental Microbiology,2005,7(11):1711-1724
40.Rodrigues Coelho M R,de Vos M,Carneiro N P,Marriel I E?,Paiva E,Seldin L.Diversity of nifH gene pools in the rhizosphere of two cultivars of sorghum(Sorghum bicolor)treated with contrasting levels of nitrogen fertilizer.FEMS Microbiology Letters,2008,279(1):15-22
41.Wang S,Gonzalez P G,Ye J,Huang D F.Abundance and diversity of nitrogen-fixing bacteria in rhizosphere and bulk paddy soil under different duration of organic management.World Journal of Microbiology&Biotechnology,2012,28(2):493-503
42.Gutiérrez-Zamora M L,Martínez-Romero E.Natural endophytic association between Rhizobium etli and maize(Zea mays L.).Journal of Biotechnology,2001,91(2-3):117-126
43.Patra A K,Abbadie L,Clays-Josserand A,Degrange V,Grayston SJ,Guillaumaud N,Loiseau P,Louault F,Mahmood S,Nazaret S,Philippot L,Poly F,Prosser J I,Le Roux X.Effects of management regime and plant species on the enzyme activity and genetic structure of N-fixing,denitrifying and nitrifying bacterial communities in grassland soils.Environmental Microbiology,2006,8(6):1005-1016
44.Orr C H,Leifert C,Cummings S P,Cooper J M.Impacts of organic and conventional crop management on diversity and activity of freeliving nitrogen fixing bacteria and total bacteria are subsidiary to temporal effects.PLoS One,2012,7(12):e52891