固氮菌株XD20对不同甘蔗品种的促生效应
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摘要
菌株XD20是从甘蔗根内分离获得的一株具有固氮活性的内生菌株。该试验对其进行16SrDNA鉴定和nifH基因扩增,并对其固氮、溶磷、产嗜铁素、产吲哚乙酸(IAA)等促生特性进行分析,然后接种于甘蔗,研究其对甘蔗的促生效应。结果表明:(1)16SrDNA序列的分析鉴定显示,该菌株属于成团泛菌。(2)nifH基因通用引物polyF/polyR扩增得到一条大小为360bp左右的基因序列,该菌的固氮酶活性约为3 187.8nmol·mL~(-1)·h~(-1)。(3)菌株XD20具有产生IAA的能力,IAA浓度可达36.71μg·mL~(-1),同时具有溶解有机磷和产生嗜铁素的能力。(4)接种XD20到不同甘蔗品种B8、ROC22和GT40的组培苗,接菌处理不同程度地提高3个品种甘蔗的株高、生物量以及叶片的叶绿素含量、硝酸还原酶活性、谷氨酰胺合成酶活性以及硝态氮的含量,并对品种B8的促生作用效果最为显著。
Strain XD20 was an endophytic strain with nitrogen fixation activity isolated from sugarcane roots.In this experiment,16 SrDNA identification and nifH gene amplification were carried out,and its growth promoting characteristics,such as nitrogen fixation,phosphorus dissolving,ferritin production and indole-acetic acid(IAA)were analyzed.Meanwhile,the growth promoting effects on sugarcane after inoculating were also studied.The results showed that:(1)strain XD20 was identified as Pantoea agglomerans by 16 SrDNA gene sequence.(2)About 360 bp was amplified by universal primer polyF/polyR of nifH gene,and the nitrogenase activity of the strain XD20 was 3 187.8 nmol·mL~(-1)·h~(-1) by using acetylene reductase method.(3)Strain XD20 had an ability to produce IAA,which the concentration of IAA reached 36.71μg·mL~(-1),and also can dissolve organophosphorus and produce ferritin.(4)Inoculating the strain XD20 to different varieties of sugarcane(B8,ROC22 and GT40),increased the plant height,plant biomass,chlorophyll content in leaves,nitrate reductase activity,glutamine synthase activity and nitrate nitrogen content of three sugarcane cultivars at varying degrees,and the promoting effect of B8 was the most significant.
Strain XD20 was an endophytic strain with nitrogen fixation activity isolated from sugarcane roots.In this experiment,16 SrDNA identification and nifH gene amplification were carried out,and its growth promoting characteristics,such as nitrogen fixation,phosphorus dissolving,ferritin production and indole-acetic acid(IAA)were analyzed.Meanwhile,the growth promoting effects on sugarcane after inoculating were also studied.The results showed that:(1)strain XD20 was identified as Pantoea agglomerans by 16 SrDNA gene sequence.(2)About 360 bp was amplified by universal primer polyF/polyR of nifH gene,and the nitrogenase activity of the strain XD20 was 3 187.8 nmol·mL~(-1)·h~(-1) by using acetylene reductase method.(3)Strain XD20 had an ability to produce IAA,which the concentration of IAA reached 36.71μg·mL~(-1),and also can dissolve organophosphorus and produce ferritin.(4)Inoculating the strain XD20 to different varieties of sugarcane(B8,ROC22 and GT40),increased the plant height,plant biomass,chlorophyll content in leaves,nitrate reductase activity,glutamine synthase activity and nitrate nitrogen content of three sugarcane cultivars at varying degrees,and the promoting effect of B8 was the most significant.
引文
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[31]范作伟,孙甜田,吴海燕.溶磷微生物肥料配方筛选及其效果验证[J].吉林农业科学,2015,40(4):34-38.FAN Z W,SUN T T,WU H Y.Screening of phosphate solubilizing microorganism fertilizer formula and verification of its effect[J].Journal of Jilin Agricultural Sciences,2015,40(4):34-38.
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[2]李杨瑞,杨丽涛,谭宏伟,等.广西甘蔗栽培技术的发展进步[J].南方农业学报,2014,45(10):1 770-1 775.LI Y R,YANG L T,TAN H W,et al.Development and progress of sugarcane farming technologies in Guangxi,China[J].Journal of Southern Agriculture,2014,45(10):1 770-1 775.
[3]杨丽涛,胡春锦,林丽,等.广西甘蔗内生固氮菌资源及其应用的相关基础研究[C].2014年中国作物学会学术年会论文摘要集,北京:中国作物学会,2014.
[4]BODDEY R M,URQUIAGA S,ALVES B J R,et al.Endophytic nitrogen fixation in sugarcane:Present knowledge and future applications[J].Plant and Soil,2003,25(2):139-149.
[5]DBEREINER J.Nitrogen fixing bacteria of the genus Beijerinck Derxin the rhizosphere of sugarcane[J].Plant and soil,1961,15:211-216.
[6]REIS V M,BALDANI J I,BALDANI V L D,et al.Biological dinitrogen fixation in gramineae and palm trees[J].Critical Reviews in Plant Sciences,2000,19:227-247.
[7]KENNEDY I R,ISLAM N.The current and potential contribution of a symbiotic nitrogen fixation to nitrogen requirements on farms[J].Australian Journal of Experimental Agriculture,2001,41:447-457.
[8]覃丽萍,黄思良,李杨瑞.甘蔗内生固氮菌的研究进展[J].中国农学通报,2005,21(5):150-152.QIN L P,HUANG S L,LI Y R.Research Progress in Endophytic diazotrop[J].Chinese Agricultural Science Bulletin,2005,21(5):150-152.
[9]FUENTES RAMIREZ L E,JIMENEZ SALGADO T,ABARCAOCAMPO I R,et al.Acetobacter diazotrophicus,an indoleacetic acid producing bacterium isolated from sugarcane cultivars of Mexico[J].Plant and Soil,1993,154:145-150.
[10]BASTIAN F,COHEN A,PICCOLI P,et al.Production of indole-3-acetic acid and gibberellins A1and A3by Acetobacter diazotrophicus and Herbaspirillum seropeicae in chemicallydefined culture media[J].Plant Growth Regulation,1998,24(1):7-11.
[11]杨海莲,孙晓璐,宋未,等.水稻内生阴沟肠杆菌MR12的鉴定及其固氮和防病作用[J].植物病理学报,2001,31(1):92-93.YANG H L,SUN X L,SONG M,et al.Studies on the rice endophytic bacteria Enterobacter cloacae MR12Sidentification and its effects of nitrogen fixation and biological control to plant disease[J].Acta Phytopathologica Sinica,2001,31(1):92-93.
[12]BRAUN K A,JACOBSEN B J,SANDS D C.Biological control of Pscudomonas syringae pv.syringae,the causal agent of basal kermel blight of barley by antagonistic Pantoea agglomerans[J].Phytopathology,2000,90(4):368-375.
[13]DURSUN A,EKINCI,M,DONMEZ M F.Effects of foliar application of plant growth promoting bacterium on chemical contents,yield and growth of tomato(Lycopersicon esculentum L.)and cucumber(Cucumis sativus L.)[J].Pak.J.Bot,2010,42,3 349-3 356.
[14]GOND S K,TORRES M S,BERGEN M S,et al.Induction of salt tolerance and up-regulation of aquaporin genes in tropical corn by rhizobac-terium Pantoea agglomerans[J].Lett Appl Microbiol,2015,60(4):392-399.
[15]FEI L,YA W,QING G Zet al.Diversity and plant growth promoting activities of the cultivable rhizo-bacteria of Dongxiang wild rice(Oryza rufipogon)[J].Biodiver Sci,2011,19,476-484.
[16]RUPPEL S,HECHT-BUCHHOLZ C,REMUS Ret al.Settle-ment of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat:an investigation using ELISA and transmission electron microscopy[J].Plant Soil,1992,145,261-273.
[17]VERMA S C,SINGH A,CHOWDHURY S P,et al.Endophytic colonization ability of two deep-water rice endophytes,Pantoea sp.and Ochrobactrum sp.using green fluorescent protein reporter[J].Biotechnol Lett,2004,26,425-429.
[18]韩梅,罗培宇,肖亦农,等.玉米内生固氮菌的分离鉴定及其促生长作用研究[J].沈阳农业大学学报,2010,41(1):94-97.HAN M,LUO P Y,XIAO Y L,et al.Isolation of endophytic N2-fixing bacteria of corn rice and their function for promoting growth[J].Journal of Shenyang Agricultural U-niversity,2010,41(1):94-97.
[19]胡春锦,林丽,史国英,等.广西甘蔗根际高效联合固氮菌的筛选及鉴定[J].生态学报,2012,32(15):4 742-4 745.HU C J,LIN L,SHI G Y,et al.Screening and identification of associative nitrogen fixation bacteria in rhizosphere of sugarcane in Guangxi[J].Acta Ecologica Sinica,2012,32(15):4 745-4 752.
[20]梁建根,竺利红,吴安吉,等.土壤中分泌嗜铁素细菌的筛选及影响其分泌因素的研究[J].园艺学报,2007,34(4):1 023-1 028.LIANG J G,ZHU L H,WU A J,et al.Studies on screening of siderophore producing bacteria in the soil and factors influencing its secretion[J].Acta Horticulturae Sinica,2007,34(4):1 023-1 028.
[21]YAO T.Associative nitrogen-fixing bacteria in the rhizosphere of Avena sativain an alpine regionⅡPhosphate-solubilizing power and auxin production[J].Acta Prataculturae Sinica,2004,13(3):85-90.
[22]魏春燕,邢永秀,莫遥,等.绿色荧光蛋白基因标记的固氮菌DX120E在甘蔗植株内的定殖[J].作物学报,2014,40(6):1 132-1 139.WEI C Y,XING Y X,MO Y,et al.Colonization of nitrogen fixing bacterial strain Klebsiella sp.DX120Elabeled with Green Fluorescent Protein(GFP)gene within sugarcane plants[J].Acta Agronomica Sinica,2014,40(6):1 132-1 139.
[23]郑炳松.现代植物生理生化研究技术[M].北京:气象出版社,2006.
[24]韦莉萍.不同钼水平对甘蔗固氮生理生化的影响[D].南宁:广西大学,2005.
[25]邹琦.植物生理学实验指南[M].北京:中国农业出版社,2000.
[26]CHEBOTAR V K,MALFANOVA N V,SHCHERBAKOVA V,et al.Endophytic bacteria in microbial preparations that improve plant development(review)[J].Appl Biochem Microbiol,2015,51(3):271-277.
[27]DASTAGER S G,DEEPA C K,PUNEET S C,et al.Isolation and characterization of plant growth-promoting strain Pantoea NII-186from Western Ghat Forest soil[J].Ind.Lett.Appl Microbiol,2009,49,20-25.
[28]ZHANG X,LI E,XIONG X,et al.Colonization of endophyte Pantoea agglomerans YS19on host rice,with formation of multicellular symplasmata[J].World.J.Microbiol.Biotechnol,2010,26,1 667-1 673.
[29]AMBREEN AHMED,SHAHIDA HASNAIN,et al.Auxinproducing Bacillus sp.:Auxin quantification and effect on the growth of Solanum tuberosum[J].2010,313-319.
[30]PEIXA A,MATEOSB P F,RODRIGUEZ B C,et al.Growth promotion of common bean(Phaseolus vulgaris L.)by a strain of Burkholderia cepacia under growth chamber conditions[J].Soil Biology&Biochemistry,2001,33:1 927-1 935.
[31]范作伟,孙甜田,吴海燕.溶磷微生物肥料配方筛选及其效果验证[J].吉林农业科学,2015,40(4):34-38.FAN Z W,SUN T T,WU H Y.Screening of phosphate solubilizing microorganism fertilizer formula and verification of its effect[J].Journal of Jilin Agricultural Sciences,2015,40(4):34-38.
[32]余贤美,郑服丛.嗜铁素在促进植物生长及病害防治等方面的应用[J].中国农学通报,2007,23(8):507-510.YU X M,ZHENG F C.The Development and utilization of siderophore on plant growth promotion and plant disease control[J].Chinese Agricultural Science Bulletin,2007,23(8):507-510.
[33]FENG Y,SHEN D,SONG W.Rice endophyte Pantoea YS19promotes host plant growth and affect allocations of host photosynthates[J].Appl.Microbial,2010,100:938-945.
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