Chinese Bulletin of Botany ›› 2018, Vol. 53 ›› Issue (4): 519-527.doi: 10.11983/CBB18007

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

An Efficient Nutrient Solution System to Study Symbiotic Nitrogen Fixation in Soybean

Ai Wenqin1, Jiang Hanyuan1, Li Xinxin2, Liao Hong2,*()   

  1. 1College of Resources and Environment, South China Agricultural University, Guangzhou 510642, China
    2Root Biology Center, Fujian Agriculture and Forestry University, Fuzhou 350002, China
  • Received:2018-01-09 Accepted:2018-02-12 Online:2018-09-11 Published:2018-07-01
  • Contact: Liao Hong
  • About author:† These authors contributed equally to this paper


To establish a nutrient solution system for soybean cultivation with effective nodulation and relatively high yield, we first evaluated the effect of nitrogen (N) content and rhizobia inoculation on soybean growth, yield and biological nitrogen fixation (BNF). Too high or too low N supply affected soybean growth, yield and N2 fixation capacity. Also, the required optimal N level was much higher for plant growth than that of BNF. Furthermore, the highest nitrogenase activity in soybean occurred before the first stage of reproduction (R1 stage). Thus, the enzyme activity could facilitate nodule formation and N2 fixation with a lower N supply before the R1 stage and a higher N supply afterward to promote plant growth and yield in soybean. A second experiment was conducted to optimize N supply (i.e., low N supply before R1 and medium N supply at the beginning of R1). As compared with continuously supplied high N, the optimal scheme promoted BNF with more and larger nodules with higher nitrogenase activity and also maintained a good growth performance with higher 100 grain weight and about sustained 80% of soybean yield. These results provide a reference for nutrient receipt for soybean BNF study as well as soybean production for high yield and nutrient efficiency.

Key words: soybean, nodule, biological nitrogen fixation, nutrient solution, yield

Figure 1

Effects of nitrogen supply on soybean nodulation(A) Nodule number; (B) Nodules fresh weight; (C) Average nodules fresh weight. V4 and R1 represent different growth stages of soybean. N1-N5 indicate 300 μmol·L-1, 900 μmol·L-1, 2 400 μmol·L-1, 4 800 μmol·L-1 and 7 200 μmol·L-1 nitrogen level, respectively. Different lowercase letters indicate significant differences among different N supply levels (P<0.05)."

Figure 2

Effects of nitrogen supply on nitrogenase activity at different growth stages of soybean(A) V4 stage; (B) R1 stage. N1-N5 see Figure 1. Different lowercase letters indicate significant differences among different N supply levels (P<0.05)."

Table 1

Effect of nitrogen supply levels and inoculation of rhizobium on soybean growth and yield"

F value Shoot dry weight Root dry weight Grain weight Hundred grain weight
N 67.15*** 134.57*** 50.75*** 36.33***
R 0.89* 2.11* 49.53*** 0.398
N×R 4.31* 14.80*** 8.31*** 4.45**

Figure 3

Effects of nitrogen supply on soybean growth(A) Up-ground dry weight; (B) Root dry weight. N1-N5 see Figure 1. Different lowercase letters indicate significant differences among different N supply levels (P<0.05)."

Figure 4

Effects of nitrogen supply levels on soybean yield(A) Pictures of soybean seeds; (B) Grain weight; (C) Hundred grain weight. N1-N5 see Figure 1. Different lowercase letters indicate significant differences among different N supply levels (P<0.05)."

Figure 5

Effects of rhizobium inoculation on soybean nodule growth and nitrogenase activity(A) Pictures of nodules; (B) Nodule number; (C) Nodule dry weight; (D) Nitrogenase activity. Different lowercase letters indicate significant differences among different growth stages of soybean (P<0.05)."

Figure 6

Soybean yield as affected by different nitrogen or rhizobia inoculation treatments(A) Pod number; (B) Grain number; (C) Grain weight; (D) Hundred grain weight. LN-R: Low N without inoculation treatment; LN+R: Low N with inoculation treatment; HN-R: High N without inoculation treatment. Different lowercase letters indicate significant differences among different treatments (P<0.05)."

1 陈文新, 陈文峰 (2004). 发挥生物固氮作用减少化学氮肥用量. 中国农业科技导报 6(6), 3-6.
2 程凤娴, 曹桂芹, 王秀荣, 赵静, 严小龙, 廖红 (2008). 华南酸性低磷土壤中大豆根瘤菌高效株系的发现及应用. 科学通报 53, 2903-2910.
3 邸伟, 金喜军, 马春梅, 龚振平, 董守坤, 张磊 (2010). 施氮水平对大豆氮素积累与产量影响的研究. 核农学报 24, 612-617.
4 胡浩南, 敖俊华, 黄晓财, 李欣欣, 廖红 (2017). 甘蔗不同组织联合固氮能力评价. 植物生理学报 53, 437-444.
5 李欣欣, 许锐能, 廖红 (2016). 大豆共生固氮在农业减肥增效中的贡献及应用潜力. 大豆科学 35, 531-535.
6 李艳, 盖钧镒 (2017). 大豆向热带地区发展的遗传基础. 植物学报 52, 389-393.
7 李宗盛, 李展辉, 邓建军 (1986). 不同时期施氮对大豆产量影响的研究. 土壤肥料 (6), 46-47.
8 罗进, 曹智 (2017). 2016年国内外大豆市场回顾及2017年展望. 中国畜牧杂志 53(4), 160-165, 178.
9 彭玉新 (2009). 施肥对大豆产量及品质的影响研究. 现代农业科技(18), 19, 21.
10 王庆胜 (2010). 根瘤菌对大豆产量及品质的影响. 黑龙江农业科学(9), 138, 147.
11 Alam F, Bhuiyan MAH, Alam SS, Waghmode TR, Kim PJ, Lee YB (2015). Effect of Rhizobium sp. BARIRGm901 ino- culation on nodulation, nitrogen fixation and yield of soybean(Glycine max) genotypes in gray terrace soil. Biosci Biotechnol Biochem 79, 1660-1668.
12 Alves BJR, Boddey RM, Urquiaga S (2003). The success of BNF in soybean in Brazil.Plant Soil 252, 1-9.
13 Brewin NJ (1991). Development of the legume root nodule.Annu Rev Cell Biol 7, 191-226.
14 Daimon H, Hori K, Shimizu A, Nakagawa M (1999). Nitrate-induced inhibition of root nodule formation and nitrogenase activity in the peanut (Arachis hypogaea L.). Plant Prod Sci 2, 81-86.
15 Fujikake H, Yamazaki A, Ohtake N, Sueyoshi K, Matsuhashi S, Ito T, Mizuniwa C, Kume T, Hashimoto S, Ishioka NS, Watanabe S, Osa A, Sekine T, Uchida H, Tsuji A, Ohyama T (2003). Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules.J Exp Bot 54, 1379-1388.
16 Gan YB, Stulen I, van Keulen H, Kuiper PJC (2004). Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW·g-1 root dry weight) and specific N2 fixation (N2 fixed·g-1 root dry weight) in soybean.Plant Soil 258, 281-292.
17 Hungria M, Campo RJ, Mendes IC (2005). Reinoculation increasing soybean grain yield in Brazil. In: Proceedings of the 14th International Nitrogen Fixation Congress. Dordrecht: Springer. pp. 315-315.
18 Hungria M, Franchini JC, Campo RJ, Crispino CC, Mor- aes JZ, Sibaldelli RNR, Mendes IC, Arihara J (2006). Nitrogen nutrition of soybean in Brazil: contributions of biological N2 fixation and N fertilizer to grain yield.Can J Plant Sci 86, 927-939.
19 Li XX, Zhao J, Tan ZY, Zeng RS, Liao H (2015). GmEXPB2, a cell wall β-expansin, affects soybean nodulation through modifying root architecture and promoting nodule formation and development.Plant Physiol 169, 2640-2653.
20 Qin L, Jiang H, Tian J, Zhao J, Liao H (2011). Rhizobia enhance acquisition of phosphorus from different sources by soybean plants.Plant Soil 349, 25-36.
21 Qin L, Zhao J, Tian J, Chen LY, Sun ZA, Guo YX, Lu X, Gu M, Xu GH, Liao H (2012). The high-affinity phosphate transporter GmPT5 regulates phosphate transport to nodu- les and nodulation in soybean.Plant Physiol 159, 1634-1643.
22 Saito A, Tanabata S, Tanabata T, Tajima S, Ueno M, Ishikawa S, Ohtake N, Sueyoshi K, Ohyama T (2014). Effect of nitrate on nodule and root growth of soybean (Glycine max (L.) Merr.). Int J Mol Sci 15, 4464-4480.
23 Tang F, Yang SM, Liu JG, Zhu HY (2016). Rj4, a gene controlling nodulation specificity in soybeans, encodes a thau- matin-like protein but not the one previously reported. Plant Physiol 170, 26-32.
24 Wang D, Yang SM, Tang F, Zhu HY (2012). Symbiosis specificity in the legume-rhizobial mutualism.Cell Microbiol 14, 334-342.
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[1] Bingyu Zhang;Xiaohua Su*;Xiangming Zhou. Gene Regulation in Flower Development in the Forest[J]. Chinese Bulletin of Botany, 2008, 25(04): 476 -482 .
[2] Cai Ji-jiong and Wang Zi-qing. A Preliminary Report on Pollen Morphology and Constituent of Pinus massoniana[J]. Chinese Bulletin of Botany, 1988, 5(03): 167 -169 .
[3] Zhang Fu-ren and Mo Ri-gen. A Simple Technique for Observing Fracture Surface of Pollen Grains by SEM[J]. Chinese Bulletin of Botany, 1992, 9(03): 63 -64 .
[4] MA Yue-Ping CHEN Fan DAI Si-Lan. Studies of LEAFY Homologue Genes in Higher Plants[J]. Chinese Bulletin of Botany, 2005, 22(05): 605 -613 .
[5] Lin Zhong-Ping. Isolating of DNA from Plant Materials[J]. Chinese Bulletin of Botany, 1984, 2(04): 44 -46 .
[6] Liu Rong-zhen and Wang Hou. The Effect of Ammonia Humic Acid Fertilizers on the Activity of End-Group Oxidase of Rice Seeding in Sludge[J]. Chinese Bulletin of Botany, 1985, 3(06): 21 -23 .
[7] . [J]. Chinese Bulletin of Botany, 1994, 11(专辑): 45 .
[8] Li Ru-juan;Shang Zong-yan and Zhang Ji-zu. The Chromosome Observation on 3 Species in the Genus Gynostemma[J]. Chinese Bulletin of Botany, 1989, 6(04): 245 -247 .
[9] Tai Wang;Qian Qian;Ming Yuan;Xiaojing Wang;Weicai Yang;Lijia Qu;Hongzhi Kong;Yinong Xu;Gaoming Jiang;Kang Chong. Research Advances on Plant Science in China in 2009[J]. Chinese Bulletin of Botany, 2010, 45(03): 265 -306 .
[10] Liang Shi-chu. A Preliminary Study on the Structure and Dynamics of Pubescent Hornbeam Population in Karst Mountain of Guiyang[J]. Chin J Plan Ecolo, 1992, 16(2): 108 -117 .