联合固氮菌叶面接种剂的优化及其在玉米叶际的定殖
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摘要
【背景】联合固氮菌由于不具有宿主专一性,在土壤、叶际中广泛存在,对生态系统氮素供应有着重要贡献,它还可以通过分泌生长激素等间接作用促进植物生长,可作为重要的农业生产菌剂。土壤接种剂由于受土著微生物的竞争和土壤抑菌物质等的影响,接种效果不稳定,难以推广使用。相比于土壤环境,叶际生境相对简单且表面积巨大,进行叶际接种是固氮菌剂推广应用的一个新思路。【目的】优化联合固氮菌菌株W12接种添加剂,制备液体接种剂并研究其在玉米叶际的定殖效果。【方法】对菌株W12进行菌落PCR测序,构建系统发育树并确定分类地位。分别在培养液中添加不同浓度梯度的羧甲基纤维素(Carboxymethyl cellulose,CMC)和甘油(Glycerol,Gly),测量菌株W12生长曲线和固氮酶活性,优化添加剂浓度并制备液体接种剂,对接种剂的有效保存时间进行检测。将接种剂喷洒到玉米叶际,测量其对玉米产量和植株含氮量的影响,并通过低氮培养基进行回收计数。【结果】固氮菌菌株W12的16S r RNA基因序列与变栖克雷伯氏菌(Klebsiella variicola)的相似性高达99%,在培养液中添加CMC和甘油对菌株W12的生长无明显促进和抑制效果,但均提高了固氮酶活性。添加甘油制备的接种剂在盆栽和大田玉米叶面喷施后,在玉米生长末期叶际回收到的W12类似菌分别为4.3×105 CFU/g叶片和1.7×105 CFU/g叶片,显著高于未接种的处理;而且大田玉米籽粒、茎部和叶片的含氮量高于不接种的对照处理。经过90 d贮藏后,4°C保存的接种剂剩余活菌数均高于1.0×108 CFU/m L。【结论】羧甲基纤维素和甘油的添加不仅有利于固氮菌液体接种剂在叶片的附着,并能显著提高联合固氮菌菌株W12的固氮酶活性,低温冷藏可保证液体接种剂的有效活菌数;液体接种剂在玉米叶际喷施后,菌株W12能够成功定殖,并显著提高玉米植株和籽粒含氮量。研究结果为固氮菌叶面接种剂的制备和应用,以及实现农业氮肥减施保产的目标提供了借鉴意义。
[Background] Associative nitrogen-fixing bacteria are non-host specific, and widely distributed in soil and phyllosphere environments, playing an important role in nitrogen supply for ecosystem. They also can promote plant growth indirectly via producing hormone for host plants or protecting plant from disease attacking, therein have great potential for agriculture application. However, the application of microbial inoculant into soil is limited o wing to the unstable efficiency in field, due to the competition from indigenous microorganisms and inhibition effect of soils. Compared to soil habitat, phyllosphere is relatively simple and constitute large areas. Foliar application of associative nitrogen fixing bacterial inoculant provides an alternative way instead of soil application in agriculture. [Objective] To optimize the liquid inoculant of an associative nitrogen-fixing bacteria strain W12 by adding different additives, and to track its coloniz ation on the phyllosphere of maize after foliar application. [Methods] Firstly, the 16 S r RNA and nif H gene of strain W12 were sequenced to identify its taxonomy. The effects of carboxymethyl cellulose(CMC) and glycerol(Gly) used as liquid inoculant additives on the growth and nitrogenase activity of strain W12 were evaluated in liquid culture and the liquid inoculant was prepared based on the optimized condition, and then the colonization of strain W12 on the phyllosphere of maize after spraying of liquid inoculant were estimated by plate counting on a low-nitrogen agar. After harvest, the grain yield and nitrogen content of grain, stem and leaf of maize in field were measured. Meanwhile, we also inspected the shelf life of inoculant under different storage conditions. [Results] The phylogeny analysis based on 16 S r RNA and nif H gene both suggested that strain W12 is closely related to the species Klebsiella variicola. CMC and glycerol addition in liquid media showed no significant influence on the growth of strain W12, but remarkably improved the nitrogenase activity by 3.7 and 24.2 times, respectively. After the foliar application of liquid inoculant via leaf spraying, the recovered strain W12-similar colonies on low-nitrogen media were up to 1.7× 105 and 4.3× 105 CFU/g leaf from maize phyllosphere in pot and field experiments, significantly higher than the control without inoculation, while the nitrogen content of grain, stem and leaf of maize in inoculation treatment were higher than those treatments without inoculation. The number of viable strain W12 decreased over the storage time while storage at 4 °C maintained much higher viable bacteria than at room temperature, with the viable bacteria number more than 1.0× 108 CFU/m L after storage at 4 °C for 90 days. [Conclusion] CMC and glycerol used as additives of liquid inoculant are favorable for bacteria adhesion on leaves and could remarkably improve nitrogenase activity of strain W12. Storage at 4 °C can sustain long shelf life of strain W12 liquid inoculation, compared with room temperature. After foliar application in field, strain W12 could successfully colonize in the phyllosphere and inoculation significantly improved the nitrogen content of maize grain and plant. Our results exhibit new insights on the preparation and application of foliar inoculant, and set a good example for field application of nitrogen fixing bacteria to reduce the nitrogen fertilizer application and stabilize crop yield.
[Background] Associative nitrogen-fixing bacteria are non-host specific, and widely distributed in soil and phyllosphere environments, playing an important role in nitrogen supply for ecosystem. They also can promote plant growth indirectly via producing hormone for host plants or protecting plant from disease attacking, therein have great potential for agriculture application. However, the application of microbial inoculant into soil is limited o wing to the unstable efficiency in field, due to the competition from indigenous microorganisms and inhibition effect of soils. Compared to soil habitat, phyllosphere is relatively simple and constitute large areas. Foliar application of associative nitrogen fixing bacterial inoculant provides an alternative way instead of soil application in agriculture. [Objective] To optimize the liquid inoculant of an associative nitrogen-fixing bacteria strain W12 by adding different additives, and to track its coloniz ation on the phyllosphere of maize after foliar application. [Methods] Firstly, the 16 S r RNA and nif H gene of strain W12 were sequenced to identify its taxonomy. The effects of carboxymethyl cellulose(CMC) and glycerol(Gly) used as liquid inoculant additives on the growth and nitrogenase activity of strain W12 were evaluated in liquid culture and the liquid inoculant was prepared based on the optimized condition, and then the colonization of strain W12 on the phyllosphere of maize after spraying of liquid inoculant were estimated by plate counting on a low-nitrogen agar. After harvest, the grain yield and nitrogen content of grain, stem and leaf of maize in field were measured. Meanwhile, we also inspected the shelf life of inoculant under different storage conditions. [Results] The phylogeny analysis based on 16 S r RNA and nif H gene both suggested that strain W12 is closely related to the species Klebsiella variicola. CMC and glycerol addition in liquid media showed no significant influence on the growth of strain W12, but remarkably improved the nitrogenase activity by 3.7 and 24.2 times, respectively. After the foliar application of liquid inoculant via leaf spraying, the recovered strain W12-similar colonies on low-nitrogen media were up to 1.7× 105 and 4.3× 105 CFU/g leaf from maize phyllosphere in pot and field experiments, significantly higher than the control without inoculation, while the nitrogen content of grain, stem and leaf of maize in inoculation treatment were higher than those treatments without inoculation. The number of viable strain W12 decreased over the storage time while storage at 4 °C maintained much higher viable bacteria than at room temperature, with the viable bacteria number more than 1.0× 108 CFU/m L after storage at 4 °C for 90 days. [Conclusion] CMC and glycerol used as additives of liquid inoculant are favorable for bacteria adhesion on leaves and could remarkably improve nitrogenase activity of strain W12. Storage at 4 °C can sustain long shelf life of strain W12 liquid inoculation, compared with room temperature. After foliar application in field, strain W12 could successfully colonize in the phyllosphere and inoculation significantly improved the nitrogen content of maize grain and plant. Our results exhibit new insights on the preparation and application of foliar inoculant, and set a good example for field application of nitrogen fixing bacteria to reduce the nitrogen fertilizer application and stabilize crop yield.
引文
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[31]Taurian T,Anzuay MS,Angelini JG,et al.Phosphate-solubilizing peanut associated bacteria:screening for plant growth-promoting activities[J].Plant and Soil,2010,329(1/2):421-431
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[2]National Bureau of Statistics of China.National data[DB/OL].[2017-06-08].http://data.stats.gov.cn/easyquery.htm?cn=C01(in Chinese)中华人民共和国国家统计局.国家数据[DB/OL].[2017-06-08].http://data.stats.gov.cn/easyquery.htm?cn=C01
[3]Zhu ZL,Chen DL.Nitrogen fertilizer use in China–contributions to food production,impacts on the environment and best management strategies[J].Nutrient Cycling in Agroecosystems,2002,63(2/3):117-127
[4]Huang GQ,Wang XX,Qian HY,et al.Negative impact of inorganic fertilizes application on agricultural environment and its countermeasures[J].Ecology and Environment,2004,13(4):656-660(in Chinese)黄国勤,王兴祥,钱海燕,等.施用化肥对农业生态环境的负面影响及对策[J].生态环境,2004,13(4):656-660
[5]Guo JH,Liu XJ,Zhang Y,et al.Significant acidification in major Chinese croplands[J].Science,2010,327(5968):1008-1010
[6]Erisman JW,Bleeker A,Galloway J,et al.Reduced nitrogen in ecology and the environment[J].Environmental Pollution,2007,150(1):140-149
[7]Hu HW,Chen DL,He JZ.Microbial regulation of terrestrial nitrous oxide formation:understanding the biological pathways for prediction of emission rates[J].FEMS Microbiology Reviews,2015,39(5):729-749
[8]Cusack DF,Silver W,Mc Dowell WH.Biological nitrogen fixation in two tropical forests:ecosystem-level patterns and effects of nitrogen fertilization[J].Ecosystems,2009,12(8):1299-1315
[9]Galloway JN,Townsend AR,Erisman JW,et al.Transformation of the nitrogen cycle:recent trends,questions,and potential solutions[J].Science,2008,320(5878):889-892
[10]Saikia SP,Jain V.Biological nitrogen fixation with non-legumes:an achievable target or a dogma?[J].Current Science,2007,92(3):317-322
[11]Kennedy IR,Choudhury ATMA,Kecskés ML.Non-symbiotic bacterial diazotrophs in crop-farming systems:can their potential for plant growth promotion be better exploited?[J].Soil Biology and Biochemistry,2004,36(8):1229-1244
[12]Zhang LM,Fang P,Zhu RQ.Recent advances in research and application of associated nitrogen-fixation with graminaceous plants[J].Chinese Journal of Applied Ecology,2004,15(9):1650-1654(in Chinese)张丼梅,方萍,朱日清.禾本科植物联合固氮研究及其应用现状展望[J].应用生态学报,2004,15(9):1650-1654
[13]Bashan Y,De-Bashan LE,Prabhu SR,et al.Advances in plant growth-promoting bacterial inoculant technology:formulations and practical perspectives(1998-2013)[J].Plant and Soil,2014,378(1/2):1-33
[14]Vacher C,Hampe A,PortéAJ,et al.The phyllosphere:microbial jungle at the plant–climate interface[J].Annual Review of Ecology,Evolution,and Systematics,2016,47:1-24
[15]Lindow SE,Brandl MT.Microbiology of the phyllosphere[J].Applied and Environmental Microbiology,2003,69(4):1875-1883
[16]Pan JG,Hu Q,Qi HY,et al.Advance in the research of phyllospheric microorganism[J].Acta Ecologica Sinica,2011,31(2):583-592(in Chinese)潘建刚,呼庆,齐鸿雁,等.叶际微生物研究迚展[J].生态学报,2011,31(2):583-592
[17]Vorholt JA.Microbial life in the phyllosphere[J].Nature Reviews Microbiology,2012,10(12):828-840
[18]Rico L,Ogaya R,Terradas J,et al.Community structures of N2-fixing bacteria associated with the phyllosphere of a Holm oak forest and their response to drought[J].Plant Biology,2014,16(3):586-593
[19]Knief C,Ramette A,Frances L,et al.Site and plant species are important determinants of the Methylobacterium community composition in the plant phyllosphere[J].The ISME Journal,2010,4(6):719-728
[20]Fürnkranz M,Wanek W,Richter A,et al.Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica[J].The ISME Journal,2008,2(5):561-570
[21]Wanek W,P?rtl K.Phyllosphere nitrogen relations:reciprocal transfer of nitrogen between epiphyllous liverworts and host plants in the understorey of a lowland tropical wet forest in Costa Rica[J].New Phytologist,2005,166(2):577-588
[22]Abril AB,Torres PA,Bucher EH.The importance of phyllosphere microbial populations in nitrogen cycling in the Chaco semi-arid woodland[J].Journal of Tropical Ecology,2005,21(1):103-107
[23]Manikandan R,Saravanakumar D,Rajendran L,et al.Standardization of liquid formulation of Pseudomonas fluorescens Pf1 for its efficacy against Fusarium wilt of tomato[J].Biological Control,2010,54(2):83-89
[24]Viji G,Uddin W,Romaine CP.Suppression of gray leaf spot(blast)of perennial ryegrass turf by Pseudomonas aeruginosa from spent mushroom substrate[J].Biological Control,2003,26(3):233-243
[25]Zhang LM.Selection and identification of an associative nitrogen-fixing bacterium strain with high efficiency and investigation of its physiological characteristics and of its inoculation effects[D].Hangzhou:Master’s Thesis of Zhejiang University,2002(in Chinese)张丼梅.高效联合固氮菌的筛选、鉴定及其生理生化特征和接种效应研究[D].杭州:浙江大学硕士学位论文,2002
[26]Suzuki MT,Giovannoni SJ.Bias caused by template annealing in the amplification of mixtures of 16S r RNA genes by PCR[J].Applied and Environmental Microbiology,1996,62(2):625-630
[27]Gaby JC,Buckley DH.A comprehensive evaluation of PCR primers to amplify the nif H gene of nitrogenase[J].PLo S One,2012,7(7):e42149
[28]Zhou Y,Qiao XW,Wang J,et al.Extraction methods of microorganisms from phyllosphere[J].Bulletin of Botanical Research,2006,26(2):233-237(in Chinese)周育,乔雄梧,王静,等.植物叶际微生物提取方法研究[J].植物研究,2006,26(2):233-237
[29]Rosenblueth M,Martínez L,Silva J,et al.Klebsiella variicola,a novel species with clinical and plant-associated isolates[J].Systematic and Applied Microbiology,2004,27(1):27-35
[30]Jha CK,Saraf M.Evaluation of multispecies plant-growth-promoting consortia for the growth promotion of Jatropha curcas L.[J].Journal of Plant Growth Regulation,2012,31(4):588-598
[31]Taurian T,Anzuay MS,Angelini JG,et al.Phosphate-solubilizing peanut associated bacteria:screening for plant growth-promoting activities[J].Plant and Soil,2010,329(1/2):421-431
[32]Gupta V,Roper MM.Protection of free-living nitrogen-fixing bacteria within the soil matrix[J].Soil and Tillage Research,2010,109(1):50-54
[33]Bandyopadhyay A,St?ckel J,Min HT,et al.High rates of photobiological H2 production by a cyanobacterium under aerobic conditions[J].Nature Communications,2010,1:139
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