Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (06): 1132-1139.doi: 10.3724/SP.J.1006.2014.01132

• RESEARCH NOTES • Previous Articles    

Colonization of Nitrogen Fixing Bacterial Strain Klebsiella sp. DX120E Labeled with Green Fluorescent Protein (GFP) Gene within Sugarcane Plants

WEI Chun-Yan1,XING Yong-Xiu1,2,*,MO Yao1,LIN Li2,YANG Li-Tao1,2,3,HU Chun-Jin3,LI Yang-Rui1,2,3,*   

  1. 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources / Agricultural College of Guangxi University, Nanning 530004, China; 2 Guangxi Academy of Agricultural Sciences / Sugarcane Research Center, Chinese Academy of Agricultural Sciences / Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture / Guangxi Key laboratory of Sugarcane Genetic Improvement, Nanning 530007, China; 3 Microbiology Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
  • Received:2013-10-23 Revised:2014-03-04 Online:2014-06-12 Published:2014-03-24

Abstract:

To explore the invasion patterns of N2-fixing bacteria strain Klebsiella sp. DX120E and its colonization in sugarcane, and to determine the optimum concentrations of inoculum, we inoculated tissue culture seedlings of two sugarcane varieties (GT21 and B8) with different concentrations of Klebsiella sp. bacteria strain DX120E labeled by green fluorescent protein (GFP) gene, and with ddH2O as CK. The results showed that the strain DX120E propagated in root, leaf sheath and leaf of sugarcane seedlings, and bacteria quantities in different parts were root > leaf sheath > leaf. There were no differences in the maximum propagation quantity under various inoculating concentrations, with the optimum concentration of 102 CFU mL–1.Fluorescence microscope observation indicated that the bacteria could invade root through the surface cracks, generating sites of main root and lateral root, and root fracture, and intensively propagate in intercellular spaces and cells of the root, also transfer to mesophyll cells and vascular bundle sheath cells in leaf.

Key words: Nitrogen fixing bacteria, Klebsiella sp., Sugarcane, Inoculation levels, Bacteria quantites, Colonization

[1] Dôbereiner J. Nitrogen- fixing bacteria of the genus Beijerinckia derxin the rhizosphere of sugar cane. Plant Soil, 1966, 15: 211–216

[2] Asis C A, Kubota M, Ohta H, Arima Y, Chebotar V K, Tsuchiya K, Alzao S. Isolation and partial characterization of endophytic diazotrophs associated with Japanese sugarcane cultivar. Soil Sci Plant Nutr, 2000, 46: 759–765

[3] Govindarajan M, Kwon S W, Weon H Y. Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9. World J Microbiol Biotechnol, 2007, 23: 997–1006

[4] 罗霆, 欧阳雪庆, 杨丽涛, 李杨瑞. 一株有固氮能力的甘蔗克雷伯氏菌的分离鉴定及固氮特性. 热带作物学报, 2010, 31: 972–978

Luo T, Ou-Yang X Q, Yang L T, Li Y R. An endophytic nitrogen-fixing bacterium Klebsiella sp. strain L03 from sugarcane: isolation, identification and characterization. Chin J Trop Crops, 2010, 31: 972–978 (in Chinese with English abstract)

[5] Taulé C, Mareque C, Barlocco C, Hackembruch F, Reis V M, Sicardi M, Battistoni F. The contribution of nitrogen fixation to sugarcane (Saccharum officinarum L.), and the identification and characterization of part of the associated diazotrophic bacterial community. Plant Soil, 2012, 356: 35–49

[6] 胡春锦, 林丽, 史国英, 汪茜, 王钱崧, 李杨瑞. 广西甘蔗根际高效联合固氮菌的筛选及鉴定. 生态学报, 2012, 32: 4745–4752

Hu C J, Lin L, Shi G Y, Wang Q, Wang Q S, Li Y R. Screening and identification of associative nitrogen fixation bacteria in rhizosphere of sugarcane in Guangxi. Acta Ecol Sin, 2012, 32: 4745–4752 (in Chinese with English abstract)

[7] Cavalcante VA, Döbereiner J. A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant Soil, 1988, 108: 23–31

[8] Cocking E C, Stone P J. Davey M R. Intracellular colonization of roots of Arabidopsis and crop plants by Gluconacetobacter diazotrophicus. In Vitro Cell Dev Plant, 2006, 42: 74–82

[9] Luna M F, Galar M L, Aprea J, Molinari M L, Boiardi J L. Colonization of sorghum and wheat by seed inoculation with Gluconacetobacter diazotrophicus. Biotechnol Lett, 2010, 32: 1071–1076

[10] Adriano-Anaya M L, Salvador-Figueroa M, OcampoJ A, García-Romera I. Hydrolytic enzyme activities in maize (Zea mays) and sorghum (Sorghum bicolor) roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices. Soil Biol & Biochem, 2006, 38: 879–886

[11] Baldani J L, Baldani V L D, Seldin L, Döbereiner J. Characterization of Herbaspirillum seropedicae gen. nov., a root associated nitrogen-fixing bacterium. Int J Syst Bacteriol, 1986, 36: 86–93

[12] Olivares F L, Baldani V L D, Reis VM, Baldani J I, Döbereiner J. Occurrence of the endophytic diazotrophs Herbaspirillum spp. in root, stems, and leaves, predominantly of gramineae. Biol Fertil Soils, 1996, 21: 197–200

[13] James E K, Olivares F L. Infection and colonization of sugarcane and other graminaceous plants by endophytic diazotrophs. Crit Rev Plant Sci, 1998, 17: 77–119

[14] James E K, Reis V M, Olivares F L, Baldani J I, Döbereiner J. Infection of sugarcane by the nitrogen-fixing bacterium Acetobacter diazotrophicus. J Exp Bot, 1994, 45: 757–766

[15] James E K, Olivares F L, Oliveira A L M, Reis F B, Silva L G. Reis V M. Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions. J Exp Bot, 2001, 52: 747–76

[16] Dong Z, Canny M J, McCully M E, Roboredo M R, Cabadilla C F, Ortega E, Rodes R. A nitrogen fixing endophyte of sugarcane stems: a new role for the apoplast. Plant Physiol, 1994, 105: 1139–1147

[17] Dong A, Heydrich M, Bernard K, McCully M E. Further evidence that N2 fixing endophytic bacterium from the intercellular spaces of sugarcane stems in Acetobacter diazotrophicus. Appl Environ Microbiol, 1995, 61: 1843–1846

[18] Dong Z, Mccully M E, Canny M J. Does Acetobacter diazotrophicus live and move in the xylem of sugarcane stems? Anatomical and physiological data. Ann Bot, 1997, 80: 147–158

[19] Haseloff J, Siemering K R. The uses of green fluorescent protein in plants. In: Chalfie M, Kain S.eds. Green Fluorescent Protein: Properties, Applications and Protocols. Hobokem, New Jersey: John Wiley&Sons. Inc., 1998. pp 191–220

[20] Singh M K, Kushwaha C, Singh R K. Studies on endophytic colonization ability of two upland rice endophytes, Rhizobium sp. and Burkholderia sp., using green fluorescent protein reporter. Curr Microbiol, 2009, 59: 240–243

[21] Lin L, Li Z G, Hu C J, Zhang X C, Chang S P, Yang L T, Li Y R, An Q L. Plant growth-promoting nitrogen-fixing enterobacteria are in association with sugarcane plants growing in Guangxi, China. Microbes Environ, 2012, 27: 391–398

[22] James E K, Gyaneshwar P, Mathan N, BarraquioW L, Reddy P M, Iannetta P P M, Olivares F L, Ladha J K. Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67. Mol Plant Microbe Interact, 2002, 15: 894–906

[23] Roncata-Maccari L D B, Ramos H J O, Pedrosa F O, Alquini Y, Chubatsu L S, Yates M G, Rigo L U, Steffens M B R, Souza E M. Endophytic Herbaspirillum seropesicae expresses nif genes in gramineous plants. FEMS Microbial Ecol, 2003, 45: 39–47

[24] Chi F, Shen S H, Cheng H P, Jing Y X,Youssef G Y, Frank B D. Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology. Appl Environ Microbiol, 2005, 71: 7271–7278

[25] Liu X M, Zhao H X, Chen S F. Colonization of maize and rice plants by strain Bacillus megaterium C4. Curr Microbiol, 2006, 52: 186–190

[26] Hurek T, Reinhold-Hurek B, Montagu M V, Kellenberger E. Root colonization and systemic spreading of Azoarcus sp. Stain BH72 in grasses. J Bacteriol, 1994, 176: 1913–1923

[27] 吕泽勋, 李久蒂, 朱至清. 用绿色荧光蛋白基因(gfp)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖. 农业生物技术学报, 2001, 9: 13–18

Lu Z X, Li J D, Zhu Z Q. Klebsiella oxytoca SG-11 labeling by green fluorescent protein gene (gfp) and its colonization in rice seedling roots. J Agric Biotechnol, 2001, 9: 13–18 (in Chinese with English abstract)

[28] 陶艳会, 林会, 赵斌. 内生固氮菌HAUM10对水稻的侵染定殖规律及促生效应. 湖北农业科学, 2012, 51: 1257–1262

Tao Y H, Lin H, Zhao B. Infection and colonization of endophytic diazotroph HAUM10 on rice seedlings and its plant growth-promoting role. Hubei Agric Sci, 2012, 51: 1257–1262 (in Chinese with English abstract)

[29] Elbeltagy A, Nishioka K, Sato T, Suzuki H, Ye B, Hamada T, Isawa T, Mitsui H, Minamisawa K. Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Appl Environ Microbiol, 2001, 67: 5285–5293

[30] Zakria M, Njoloma J, Saeki Y, Akao S. Colonization and nitrogen-fixing ability of Herbaspirillum sp. Strain B501 gfp1 and assessment of its growth-promoting ability in cultivates rice. Microbes Environ, 2007, 22: 197–206

[31] Njoloma J,Tanaka K, Shimizu T, Nishiguchi T, Zakria M, Akashi R, Oota M, Akao S. Infection and colonization of aseptically micropropagated sugarcane seedling by nitrogen-fixing endophytic bacterium, Herbaspirillum sp. B501 gfp1. Biol Fertil Soils, 2006, 43: 137–143

[32] Liu Y, Chen S F, Li J L. Colonization pattern of Azospirillum brasilense Yu 62 on maize roots. Acta Bot Sin, 2003, 45: 748–752

[33] Reis V M, Olivares F L, Oliveira A L M, Reis F B, Baldani J I, Döbereiner J. Technical approaches to inoculate micropropagated sugar cane plants with Acetobacter diazotrophicus. Plant Soil, 1999, 206: 205–211

[34] Suman A, Gaur A, Shrivastava A K, Yadav R L. Improving sugarcane growth and nutrient uptake by inoculating Gluconacetobacter diazotrophicus. Plant Growth Regul, 2005, 47: 155–162

[35] 罗霆, 欧阳雪庆, 杨丽涛, 李杨瑞. 15N同位素稀释法研究固氮菌接种对甘蔗生物固氮的影响. 核农学报, 2010, 24: 1026–1031

Luo T, Ou-Yang X Q, Yang L T, Li Y R. Effect of nitrogen-fixing bacteria inoculation on biological nitrogen fixation in sugarcane by 15N isotope dilution technique. J Nucl Agr Sci , 2010, 24: 1026–1031 (in Chinese with English abstract)

[36] Iniguez A L, Dong Y, Triplett E W. Nitrogen fixation in wheat provided by Klebsiella pneumonia 342. Mol Plant Microbe Interact, 2004, 17: 1078–1085

[1] XIAO Jian, CHEN Si-Yu, SUN Yan, YANG Shang-Dong, TAN Hong-Wei. Characteristics of endophytic bacterial community structure in roots of sugarcane under different fertilizer applications [J]. Acta Agronomica Sinica, 2022, 48(5): 1222-1234.
[2] ZHOU Hui-Wen, QIU Li-Hang, HUANG Xing, LI Qiang, CHEN Rong-Fa, FAN Ye-Geng, LUO Han-Min, YAN Hai-Feng, WENG Meng-Ling, ZHOU Zhong-Feng, WU Jian-Ming. Cloning and functional analysis of ScGA20ox1 gibberellin oxidase gene in sugarcane [J]. Acta Agronomica Sinica, 2022, 48(4): 1017-1026.
[3] KONG Chui-Bao, PANG Zi-Qin, ZHANG Cai-Fang, LIU Qiang, HU Chao-Hua, XIAO Yi-Jie, YUAN Zhao-Nian. Effects of arbuscular mycorrhizal fungi on sugarcane growth and nutrient- related gene co-expression network under different fertilization levels [J]. Acta Agronomica Sinica, 2022, 48(4): 860-872.
[4] YANG Zong-Tao, LIU Shu-Xian, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane ubiquitin-like protein UBL5 responses to SCMV infection and interacts with SCMV-6K2 [J]. Acta Agronomica Sinica, 2022, 48(2): 332-341.
[5] ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, LIU Shu-Xian, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane PsbR subunit response to SCMV infection and its interaction with SCMV-6K2 [J]. Acta Agronomica Sinica, 2021, 47(8): 1522-1530.
[6] SU Ya-Chun, LI Cong-Na, SU Wei-Hua, YOU Chui-Huai, CEN Guang-Li, ZHANG Chang, REN Yong-Juan, QUE You-Xiong. Identification of thaumatin-like protein family in Saccharum spontaneum and functional analysis of its homologous gene in sugarcane cultivar [J]. Acta Agronomica Sinica, 2021, 47(7): 1275-1296.
[7] WANG Heng-Bo, CHEN Shu-Qi, GUO Jin-Long, QUE You-Xiong. Molecular detection of G1 marker for orange rust resistance and analysis of candidate resistance WAK gene in sugarcane [J]. Acta Agronomica Sinica, 2021, 47(4): 577-586.
[8] ZHANG Rong-Yue, WANG Xiao-Yan, YANG Kun, SHAN Hong-Li, CANG Xiao-Yan, LI Jie, WANG Chang-Mi, YIN Jiong, LUO Zhi-Ming, LI Wen-Feng, HUANG Ying-Kun. Identification of brown rust resistance and molecular detection of Bru1 gene in new and main cultivated sugarcane varieties [J]. Acta Agronomica Sinica, 2021, 47(2): 376-382.
[9] CANG Xiao-Yan, XIA Hong-Ming, LI Wen-Feng, WANG Xiao-Yan, SHAN Hong-Li, WANG Chang-Mi, LI Jie, ZHANG Rong-Yue, HUANG Ying-Kun. Evaluation of natural resistance to smut in elite sugarcane varieties (lines) [J]. Acta Agronomica Sinica, 2021, 47(11): 2290-2296.
[10] ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, WANG Tong, LIU Shu-Xian, SHANG He-Yang, ZHAO He, XU Jing-Sheng. Cloning of sugarcane ScCRT1 gene and its response to SCMV infection [J]. Acta Agronomica Sinica, 2021, 47(1): 94-103.
[11] ZHENG Qing-Lei,YU Chen-Jing,YAO Kun-Cun,HUANG Ning,QUE You-Xiong,LING Hui,XU Li-Ping. Cloning and expression analysis of sugarcane Fe/S precursor protein gene ScPetC [J]. Acta Agronomica Sinica, 2020, 46(6): 844-857.
[12] LUO Jun,LIN Zhao-Li,LI Shi-Yan,QUE You-Xiong,ZHANG Cai-Fang,YANG Zai-Qi,YAO Kun-Cun,FENG Jing-Fang,CHEN Jian-Feng,ZHANG Hua. Effects of different soil improvement measures on soil physicochemical properties and microbial community structures in mechanically compacted acidified sugarcane field [J]. Acta Agronomica Sinica, 2020, 46(4): 596-613.
[13] WANG Heng-Bo,QI Shu-Ting,CHEN Shu-Qi,GUO Jin-Long,QUE You-Xiong. Development and application of SSR loci in monoploid reference genome of sugarcane cultivar [J]. Acta Agronomica Sinica, 2020, 46(4): 631-642.
[14] ZHANG Hai, LIU Shu-Xian, YANG Zong-Tao, WANG Tong, CHENG Guang-Yuan, SHANG He-Yang, XU Jing-Sheng. Sugarcane PsbS subunit response to Sugarcane mosaic virus infection and its interaction with 6K2 protein [J]. Acta Agronomica Sinica, 2020, 46(11): 1722-1733.
[15] GAO Shi-Wu,FU Zhi-Wei,CHEN Yun,LIN Zhao-Li,XU Li-Ping,GUO Jin-Long. Cloning and expression analysis of metallothionein family genes in response to heavy metal stress in sugarcane (Saccharum officinarum L.) [J]. Acta Agronomica Sinica, 2020, 46(02): 166-178.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!