小麦内生固氮菌分离及其ACC脱氨酶测定

doi:10.3864/j.issn.0578-1752.2012.06.004

摘要/Abstract

摘要： 【目的】了解小麦内生固氮菌数量，筛选具有ACC（1-aminocyclopropane-1-carboxylate, 1-氨基环丙烷-1-羧酸）脱氨酶活性的小麦内生固氮菌，确定筛选菌株的系统发育地位与分类地位，为微生物肥料生产收集菌种资源。【方法】样品表面灭菌后采用无氮培养法筛选内生固氮菌，乙炔还原法测定菌株固氮酶活性；采用ACC唯一氮源法筛选ACC脱氨酶阳性菌，比色法定量测定ACC脱氨酶活性；PCR扩增得到菌株16S rDNA，通过序列测定和相似性分析研究菌株的系统发育；通过形态、生理生化特征和16S rDNA序列比对鉴定菌种。【结果】小麦体内固氮菌数量为（0.2—17.8）×105 cfu•g-1鲜重；分离到小麦内生固氮菌60株，固氮酶活性在1—36 nmol C2H4/h•mg蛋白，其中9株具有ACC脱氨酶活性，活性在0.87—9.32 µmol α-丁酮酸/h•mg蛋白；新分离菌株9136固氮酶活性为1.82 nmol C2H4/h•mg蛋白，ACC脱氨酶活性为9.32 µmol α-丁酮酸/h•mg蛋白，初步鉴定为假单胞菌Pseudomonas sp.。【结论】田间自然生长的小麦体内有大量固氮菌，数量在105 cfu•g-1鲜重，其中部分菌株具有ACC脱氨酶活性，个别菌株具有较高的ACC脱氨酶活性，可能对作物抵御不良环境具有作用。

关键词: 小麦, 内生固氮菌, ACC脱氨酶, 根际促生细菌

Abstract: 【Objective】 The objective of this study is to determine the ammount of wheat endophytic diazotrophs and screen for ACC (1-aminocyclopropane-1-carboxylate) deaminase activity from the diazotrophs, determine the phylogenetic and classific position of selected strains and prepare strains for microbial fertilizer production. 【Method】 Surface sterilization and nitrogen-free medium were used to isolate diazotroph and ACC was used as sole nitrogen source to screen strains with ACC deaminase activity. Nitrogenase activity was determined with acetylene reduction assay. 16S rDNA was amplified with PCR and analysed with MEGA software. Strain identification was carried out based on the morphology, physiology, biochemical test results and 16S rDNA analysis. 【Result】 The ammount of endophytic diazotrophs at jointing stage of wheat was (0.2-17.8)×105 cfu•g-1 fresh weight. Sixty endophytic diazotrophs with nitrogenase activity ranging 1-36 nmol C2H4/h•mg protein were isolated from wheat. Nine of the 60 endophytic diazotrophs were ACC deaminase positive, the range of enzyme activity is 0.87-9.32 µmol α-ketobutryric acid/h•mg protein. New isolate 9136 with nitrogenase activity 1.82 nmol C2H4/h•mg protein and ACC deaminase activity 9.32 µmol α-ketobutryric acid/h•mg protein was identified as Pseudomonas sp.. 【Conclusion】 About 105 cfu•g-1 (fresh weight) endophytic diazotrophs naturally colonized field grown wheat, some of these endophytic diazotrophs could produce ACC deaminase. A few strains showed relatively high ACC deaminase activity, and they might play a role in crop resistance to enviromental stress.

Key words: wheat, endophytic diazotrophs, ACC deaminase, PGPB

秦宝军, 罗琼, 高淼, 胡海燕, 徐晶, 周义清, 孙建光. 小麦内生固氮菌分离及其ACC脱氨酶测定[J]. 中国农业科学, 2012, 45(6): 1066-1073.

QIN Bao-Jun, LUO Qiong, GAO Miao, HU Hai-Yan, XU Jing, ZHOU Yi-Qing, SUN Jian-Guang. Isolation of Wheat Endophytic Diazotrophs and Determination of 1-Aminocyclopropane-1-Carboxylate Deaminase[J]. Scientia Agricultura Sinica, 2012, 45(6): 1066-1073.

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参考文献

[1]Barraquio W, Revilla L, Ladha J. Isolation of endophytic diazotrophic bacteria from wetland rice. Plant and Soil, 1997, 194: 15-24.

[2]Zhang G X, Peng G X, Wang E T, Yan H, Yuan Q H, Zhang W, Lou X, Wu H, Tan Z Y. Diverse endophytic nitrogen-fixing bacteria isolated from wild rice Oryza rufipogon and description of Phytobacter diazotrophicus gen. nov. sp. nov.. Archives of Microbiology, 2008, 189: 431-439.

[3]Govindarajan M, Balandreau J, Kwon S W, Weon H Y, Lakshminarasimhan C. Effects of the inoculation of Burkholderia vietnamensis and related endophytic diazotrophic bacteria on grain yield of rice. Microbial Ecology, 2008, 55(1): 21-37.

[4]Boddey R M, Urquiaga S, Alves B J R, Reis V. Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant and Soil, 2003, 252: 139-149.

[5]Govindarajan M, Balandreau J, Muthukumarasamy R, Revathi G, Lakshminarasimhan C. Improved yield of micropropagated sugarcane following inoculation by endophytic Burkholderia vietnamiensis. Plant and Soil, 2006, 280: 239-252.

[6]Shaharoona B, Arshad M, Zahir Z A. Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on nodulation in mung bean (Vigna radiata L.). Letters in Applied Microbiology, 2006, 42: 155-159.

[7]Belimov A A, Dodd I C, Safronova V I, Hontzeas N, Davies W J. Pseudomonas brassicacearum strain Am3 containing 1- aminocyclopropane-1-carboxylate deaminase can show both pathogenic and growth-promoting properties in its interaction with tomato. Journal of Experimental Botany, 2007, 58(6): 1485-1495.

[8]Jalili F, Khavazi K, Pazira E, Nejati A, Rahmani H A, Sadaghiani H R, Miransari M. Isolation and characterization of ACC deaminase- producing fluorescent pseudomonas, to alleviate salinity stress on canola (Brassica napus L.) growth. Journal of Plant Physiology, 2009, 166: 667-674.

[9]李倍金, 罗明, 周俊, 孔德江, 张铁明. 几种禾草内生固氮菌的分离及固氮活性测定. 草业学报, 2008, 17(5): 37-42.

Li B J, Luo M, Zhou J, Kong D J, Zhang T M. Isolation of endophytic diazotrophic bacteria from several gramineae grasses and determination of their nitrogenase activity. Acta Prataculturae Sinica, 2008, 17(5): 37-42. (in Chinese)

[10]Tan Z Y, Peng G X, Xu P Z, Ai S Y, Tang S H, Zhang G X, Zeng F Y. Diversity and high nitrogenase activity of endophytic diazotrophs isolated from Oryza rufipogon Griff. Chinese Science Bulletin, 2009, 54: 2839-2848.

[11]孙建光, 张燕春, 徐晶, 胡海燕. 高效固氮芽孢杆菌选育及其生物学特性研究. 中国农业科学, 2009, 42(6): 2043-2051.

Sun J G, Zhang Y C, Xu J, Hu H Y. Isolation and biological characteristic investigation on efficient nitrogen-fixing bacilli. Scientia Agricultura Sinica, 2009, 42(6): 2043-2051. (in Chinese)

[12]Penrose D M, Glick B R. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiological Plantarum, 2003, 118: 10-15.

[13]孙建光, 徐晶, 胡海燕, 张燕春, 刘君, 王文博, 孙燕华. 中国十三省市土壤中非共生固氮微生物菌种资源研究. 植物营养与肥料学报, 2009, 15(6): 1450-1465.

Sun J G, Xu J, Hu H Y, Zhang Y C, Liu J, Wang W B, Sun Y H. Collection and investigation on asymbiotic nitrogen-fixing microbial resources from 13 provinces over China. Plant Nutrition and Fertilizer Science, 2009, 15(6): 1450-1465. (in Chinese)

[14]Honma M, Shimomura T. Metabolism of 1-aminocyclopropane-1- carboxylic acid. Agricultural and Biological Chemistry, 1978, 42(10): 1825-1831.

[15]东秀珠, 蔡妙英. 常见细菌系统鉴定手册. 北京: 科学出版社, 2001.

Dong X Z, Cai M Y. Manual of Systermatic Determinative Bacteriology. Beijing: Science Press, 2001. (in Chinese)

[16]沈萍, 范秀容, 李广武. 微生物学实验. 第三版. 北京: 高等教育出版社, 1999.

Shen P, Fan X R, Li G W. Microbiology Experiment. 3rd ed. Beijing: High Education Press, 1999. (in Chinese)

[17]Ivanova E P, Christen R, Bizet C, Clermont D, Motreff L, Bouchier C, Zhukova N V, Crawford R J, Kiprianova E A. Pseudomonas brassicacearum subsp. neoaurantiaca subsp. nov., orange-pigmented bacteria isolated from soil and the rhizosphere of agricultural plants. International Journal of Systematic and Evolutionary Microbiology, 2009, 59: 2476-2481.

[18]Achouak W, Sutra L, Heulin T, Meyer J M, Fromin N, Degraeve S, Christen R, Gardan L. Pseudomonas brassicacearum sp. nov. and Pseudomonas thivervalensis sp. nov., two root-associated bacteria isolated from Brassica napus and Arabidopsis thaliana. International Journal of Systematic and Evolutionary Microbiology, 2000, 50: 9-18.

[19]Sikorski J, Stackebrandt E, Wackernagel W. Pseudomonas kilonensis sp. nov., a bacterium isolated from agricultural soil. International Journal of Systematic and Evolutionary Microbiology, 2001, 51: 1549-1555.

[20]Holt J G. Bergey's Manual of Systematic Bacteriology. 1st ed. Baltimore: Williams & Wilkins, 1984-1989.

[21]陈文新. 生物固氮//中国土壤学会. 氮素循环与农业和环境学术研讨会论文集, 厦门: 厦门大学出版社, 2001: 4-5.

Chen W X. Biological nitrogen fixation//Soil Science Society of China. Proceeding of Conference on Nitrogen Cycling and Agriculre Environment, Xiamen: Xiamen University Press, 2001: 4-5. (in Chinese)

[22]Munoz-Rojas J, Caballero-Mellado J. Population dynamics of Gluconacetobacter diazotrophicus in sugarcane cultivars and its effect on plant growth. Microbial Ecology, 2003, 46: 454-464.

[23]Iniguez A L, Dong Y, Triplett E W. Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342. Molecular Plant-Microbe Interactions, 2004, 17(10): 1078-1085.

[24]Saleem M, Arshad M, Hussain S, Bhatti A S. Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agricuture. Journal of Industrial Microbiology and Biotechnology, 2007, 34: 635-648.

[25]Arshad M, Saleem M, Hussain S. Perspectives of bacterial ACC deaminase in phytoremediation. TRENDS in Biotechnology, 2007, 25(8): 356-362.

[26]Grichko V P, Glick B R. Amelioration of flooding stress by ACC deaminase-containing plant growth-promoting bacteria. Plant Physiology and Biochemistry, 2001, 39: 11-17.

[27]Shaharoona B, Arshad M, Zahir Z A, Khalid A. Performance of Pseudomonas spp. containing ACC-deaminase for improving growth and yield of maize (Zea mays L.) in the presence of nitrogenous fertilizer. Soil Biology Biochemistry, 2006, 38: 2971-2975.

[28]Onofre-Lemus J, Hernandez-Lucas I, Girard L, Caballero-Mellado J. ACC (1-aminocyclopropane-1-carboxylate) deaminase activity, a widespread trait in Burkholderia species, and its growth-promoting effect on tomato plants. Applied and Enviromental Microbiology, 2009, 75(20): 6581-6590.