Evaluation of Nodulation Speed by Sinorhizobium Strains

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Molecular Plant Microbe Interactions
Apr 2014



Rhizobia interact symbiotically with legumes to form root nodules, where by rhizobia fix atmospheric dinitrogen into ammonia in exchange for carbon produced via photosynthesis. The symbiotic interaction is agriculturally important by reducing the need for fertilizer containing nitrogen. The root and stem nodule bacteria commonly include bacteria in the genera Rhizobium, Mesorhizobium, Sinorhizobium (Ensifer), and Bradyrhizobium, although other genera of bacteria have now been shown to form root nodule symbioses with several legume species (Weir, 2012). Different rhizobial strains form different numbers of nodules on specific legume plant varieties (or cultivars), and the nitrogen fixing effectiveness of each rhizobial strain, its ability to fix nitrogen and transfer it to the plant, is also highly variable (Toro, 1996). Some native rhizobia are ineffective at fixing nitrogen yet form a majority of nodules in filed grown plants. This is referred to as the competition for nodulation problem (Triplett and Sadowsky, 1992). Competition studies are not feasible when evaluating a large number of different native strains. However, nodulation speed of individual strains correlates well with overall competiveness and can be used to identify native strains that overcome the competition problem (De Oliveira and Graham, 1990; Bhuvaneswari et al., 1980).

Materials and Reagents

  1. Sinorhizobium meliloti strains
  2. Medicago truncatula seeds
  3. Sodium chloride (MACRON CHEMICAL, catalog number: 7581-06 )
  4. Sodium hypochlorite
  5. Ethanol (Decon Labs, catalog number: 2701 )
  6. CYG growth pouches (Mega International Inc.)
  7. Concentrated sulfuric acid (Thermo Fisher Scientific, catalog number: A300-212 )
  8. Tryptone (BD Bioscience, catalog number: 211699 )
  9. Yeast extract (Thermo Fisher Scientific, catalog number: DF0127071 )
  10. CaCl2.6 H2O (Thermo Fisher Scientific, catalog number: C79-3 )
  11. Agar (Sigma-Aldrich, catalog number: A1296 )
  12. CaCl2.6 H2O (Thermo Fisher Scientific, catalog number: C79-3 )
  13. MgSO4.7 H2O (JT Baker, catalog number: 2504 )
  14. KH2PO4 (JT Baker, catalog number: 3246-01 )
  15. Na2HPO4.2 H2O (Sigma-Aldrich, catalog number: S9763 )
  16. Fe-citrate (Sigma-Aldrich, catalog number: F3388 )
  17. MnCl2.4 H2O (Thermo Fisher Scientific, catalog number: M33-500 )
  18. CuSo4.5 H2O (Thermo Fisher Scientific, catalog number: C-493 )
  19. ZnCl2 (JT Baker, catalog number: 4326-01 )
  20. H3BO3 (Mallinckrodt, catalog number: 2549 )
  21. Na2MoO4.2 H2O (Sigma-Aldrich, catalog number: S6646 )
  22. TY medium (see Recipes)
  23. Fahraeus medium (see Recipes)


  1. P5 filter paper (Thermo Fisher Scientific, catalog number: 09-801C )
  2. Aluminum foil
  3. Plant growth chamber


  1. Medicago seed sterilization
    1. Place the seeds in a 15 ml plastic falcon tube. Add 3 ml of concentrated sulfuric acid for 5-8 min with occasional mixing.
    2. Rapidly remove the acid with pipet, and wash 4 times with sterilized water.
    3. Soak the seeds in 10% sodium hypochlorite for 90 sec.
    4. Rinse 8 times with sterile water.
    5. Place the seeds on sterile filter paper disk in a petri dish and soak with 2 ml of sterile water.
    6. Place the seeds in the refrigerator at 4 °C (in the dark) for 3- 5 days.
    7. Move seeds to room temperature and incubate (in the dark) for 1 day to pre-germinate (1-1.5 cm root length).

  2. Sinorhizobium strains
    1. Sinorhizobium meliloti strains are grown in TY medium with 0.1 % initial inoculum to the exponential growth state (~ 2 days) at 30 °C, with shaking (200 rpm).
    2. Centrifuge the culture at 8,000 x g for 10 min and wash once with sterile 0.85% NaCl.
    3. The culture should be diluted to 107 cells/ml or an OD600 0.7 with sterile 0.85% NaCl.

  3. Plant growth conditions and inoculation
    1. Plant growth pouches should be wetted with water and sterilize at 121 °C for 15 min.
    2. Add 2-4 pregerminated seedlings from the step A7 per pouch, with plant roots protruding through holes in the paper pouch tray.
    3. Add 6 ml fahraeus medium.
    4. Grow pouches in growth chamber at 25 °C with 16 h light conditions and 21 °C for 8 h dark conditions. Humidity should be between 50-70%. Light conditions should be 200-350 µmol/m2/s.
    5. Cover the roots with aluminum foil and place pouches in a container.
    6. Inoculate seedlings in pouches after 1 day with 1.0 ml of suspended culture immediately after step 10 using sterile pipette.
    7. Mark the location of the root tip on the surface of the growth pouch using a sharpie marker.

  4. Nodule analysis
    1. Examine pouches each day for the presence and number of nodules on each plant. Record the date when the first nodule formed and the number of nodules daily. Plot the number of plants nodulated (Y-axis) by days at the end of experiment.
    2. After three weeks of growth remove the pouches from the growth chamber.
    3. Count the nodules and measure the distance (in mm) that each nodule is relative to the root mark at the time of inoculation. Nodules above the root tip mark are designated as + mm and those below the root tip mark as - mm. Those at the root tip mark are designated as 0 mm. Plot the number of nodules (Y-axis) versus distance from root tip mark in a histogram. Those rhizobia with the majority of nodules above the root tip mark formed nodules earlier than those below the mark and are thought to be more competitive for nodulation.

Representative data

Figure 1. Nodulated Medicago truncatula with Sinorhizobium meliloti, after 2 weeks of growth. The plants were monitored daily and marked newly formed nodules on outer plastic cover. Horizontal lines are the root tips when the culture was inoculated. For example, the nodule marked as B3 in the middle plant is a nodule above the root tip mark as+ mm and the nodule B1 and B2 are those below the root tip mark as - mm.

Figure 2. Top view of growth pouch experiment of Medicago truncatula with tin foil lined container


  1. TY medium
    5 g tryptone
    3 g yeast extract
    1.3 g CaCl2.6 H2O or 0.87 g CaCl2.2 H2O
    Bring to 1 L with distilled H2O, adjust pH to 6.9 and sterilize by autoclaving for 30 min
    For the plate, 1.5 % agar is added
  2. Fahraeus medium (Vincent, 1970)
    0.132 g/L CaCl2
    0.120 g/L MgSO4.7 H2O
    0.100 g/L KH2PO4
    0.075 g/L Na2HPO4.2 H2O
    5 mg/L Fe-citrate
    0.07 mg/L each of MnCl2.4 H2O, CuSo4.5 H2O, ZnCl2, H3BO3, and Na2MoO4.2 H2O
    Adjust pH to 7.5 before autoclaving for 1 h for 10 L


This study was supported by grant 1237993 form The National Science Foundation.


  1. De Oliveira, L. and Graham, P. (1990). Speed of nodulation and competitive ability among strains of Rhizobium leguminosarum bv phaseoli. Arch Microbiol 153(4): 311-315.
  2. Bhuvaneswari, T. V., Turgeon, B. G. and Bauer, W. D. (1980). Early events in the infection of soybean (Glycine max L. Merr) by Rhizobium japonicum: I. Localization of infectible root cells. Plant Physiol 66(6): 1027-1031.
  3. Toro, A. (1996). Nodulation competitiveness in the Rhizobium-legume symbiosis. World J Microbiol Biotechnol 12(2): 157-162.
  4. Triplett, E. W. and Sadowsky, M. J. (1992). Genetics of competition for nodulation of legumes. Annual Reviews in Microbiology 46(1): 399-422.
  5. Vincent, J. M. (1970). A manual for the practical study of the root-nodule bacteria. A manual for the practical study of the root-nodule bacteria.
  6. Weir, B. S. (2012). The current taxonomy of rhizobia. NZ Rhizobia website.


根瘤菌与豆科植物共生相互作用形成根瘤,其中根瘤菌将大气氮固定为氨,以交换通过光合作用产生的碳。共生相互作用在农业上是重要的,通过减少对含氮肥的需要。根和茎结节细菌通常包括根瘤菌属,中生根瘤菌,中华根瘤菌属( Ensifer )和 Bradyrhizobium >,虽然其他属的细菌现在已经显示形成根瘤与多种豆科物种的共生(Weir,2012)。不同的根瘤菌菌株在特定豆科植物品种(或品种)上形成不同数目的结节,并且每种根瘤菌菌株的氮固定效率,其固定氮并将其转移到植物的能力也是高度可变的(Toro,1996)。一些天然根瘤菌在固氮但在所生长的植物中形成大多数结节方面无效。这被称为结瘤问题的竞争(Triplett和Sadowsky,1992)。当评估大量不同的天然菌株时,竞争研究是不可行的。然而,单个菌株的结瘤速度与总体竞争性良好相关,并且可用于鉴定克服竞争问题的天然菌株(De Oliveira和Graham,1990; Bhuvaneswari等人,1980)。

  • Wolff,AC,Hammond,ME,Hicks,DG,Dowsett,M.,McShane,LM,Allison,KH,Allred,DC,Bartlett,JM,Bilous,M.,Fitzgibbons,P.,Hanna,W.,Jenkins, RB,Mangu,PB,Paik,S.,Perez,EA,Press,MF,Spears,PA,Vance,GH,Viale,G.,Hayes,DF,American Society of Clinical, (2013年)。 关于乳腺癌中人表皮生长因子受体2检测的建议:美国临床肿瘤学会/学院美国病理学家临床实践指南更新。 J Clin Oncol 31(31):3997-4013。
  • Wolff,AC,Hammond,ME,Schwartz,JN,Hagerty,KL,Allred,DC,Cote,RJ,Dowsett,M.,Fitzgibbons,PL,Hanna,WM,Langer,A.,McShane,LM,Paik, ,Pegram,MD,Perez,EA,Press,MF,Rhodes,A.,Sturgeon,C.,Taube,SE,Tubbs,R.,Vance,GH,van de Vijver,M.,Wheeler,TM,Hayes,DF ,美国临床协会,O。和美国学院,P。(2007)。 美国临床肿瘤学会/美国病理学家大学关于人表皮生长因子受体2检测的指南建议乳腺癌。 25(1):118-145
  • ...
  • CYG growth pouches (Mega International Inc.)
  • Concentrated sulfuric acid (Thermo Fisher Scientific, catalog number: A300-212)
  • Tryptone (BD Bioscience, catalog number: 211699)
  • Yeast extract (Thermo Fisher Scientific, catalog number: DF0127071)
  • CaCl2.6 H2O (Thermo Fisher Scientific, catalog number: C79-3)
  • Agar (Sigma-Aldrich, catalog number: A1296)
  • CaCl2.6 H2O (Thermo Fisher Scientific, catalog number: C79-3)
  • MgSO4.7 H2O (JT Baker, catalog number: 2504)
  • KH2PO4 (JT Baker, catalog number: 3246-01)
  • Na2HPO4.2 H2O (Sigma-Aldrich, catalog number: S9763)
  • Fe-citrate (Sigma-Aldrich, catalog number: F3388)
  • MnCl2.4 H2O (Thermo Fisher Scientific, catalog number: M33-500)
  • CuSo4.5 H2O (Thermo Fisher Scientific, catalog number: C-493)
  • ZnCl 2(JTBaker,目录号:4326-01)

  • (Mallinckrodt,目录号:2549)
  • (Sigma-Aldrich,目录号:S6646),制备标题化合物,其中R 1,R 2,R 3,R 4, >
  • TY介质(参见配方)
  • Fahraeus培养基(见配方)
  • 设备

    1. P5滤纸(Thermo Fisher Scientific,目录号:09-801C)
    2. 铝箔
    3. 植物生长室


    1. 苜蓿种子灭菌
      1. 将种子放在15毫升塑料猎鹰管中。 偶尔混合,加入3ml浓硫酸5-8分钟
      2. 用移液管迅速除去酸,用无菌水洗涤4次
      3. 将种子浸泡在10%次氯酸钠中90秒
      4. 用无菌水冲洗8次
      5. 将种子放在培养皿中的无菌滤纸盘上,并用2ml无菌水浸泡
      6. 将种子放在冰箱中,在4°C(黑暗),3 - 5天
      7. 将种子移至室温并孵育(在黑暗中)1天以预发芽(1-1.5厘米根长)。

    2. 中华根瘤菌菌株
      1. 将中华根瘤菌(Sinorhizobium meliloti)菌株在含有0.1%初始的TY培养基中生长   在30℃下接种至指数生长状态(〜2天) 摇动(200rpm)
      2. 将培养物以8000xg离心10分钟,并用无菌的0.85%NaCl洗涤一次。
      3. 用无菌的0.85%NaCl将培养物稀释至10 7个细胞/ml或OD 600±0.7。

    3. 植物生长条件和接种
      1. 植物生长袋应用水润湿,并在121℃下灭菌15分钟
      2. 每个袋加入来自步骤A7的2-4个预发芽的幼苗 植物根通过纸袋托盘中的孔突出。
      3. 加入6ml fahraeus培养基。
      4. 在25℃,16小时光照条件下,在生长室中生长小袋 和21℃下8小时黑暗条件。 湿度应在50-70%之间。 光条件应为200-350μmol/m 2/s/s
      5. 用铝箔覆盖根部,将小袋放入容器中。
      6. 1天后用1.0ml悬浮液接种小袋中的幼苗   使用无菌移液管在步骤10后立即培养
      7. 使用尖角标记标记根尖在生长袋表面上的位置。

    4. 结节分析
      1. 每天检查袋每个结节的存在和数量 厂。 记录第一个结节形成的日期和数量 结节每日。 绘制结节的植物数量(Y轴)以天为单位 实验结束。
      2. 生长三周后,从生长室中取出小袋。
      3. 计数结节,并测量每个结节的距离(以mm为单位)   是相对于接种时的根标记。 以上结节 根尖尖标记被指定为+ mm,而根尖以下 标记为 - mm。 那些在根尖标记被指定为0mm。 情节 结节的数量(Y轴)与距根尖标记的距离a 柱状图。 那些根瘤菌与大多数根瘤上方的结节 尖端标记形成的结节早于那些低于标记的那些 认为对结瘤更有竞争力。


    图1.在生长2周后,用苜蓿中华根瘤菌结瘤的 。每天监测植物并在外塑料上标记新形成的结节 盖。 水平线是接种培养物时的根尖。 例如,中间植物中标记为B3的结节是根尖尖标记之上的结节为+ mm,结节B1和B2是根尖尖标记之下的结节为-mm。



    1. TY介质
      1.3g CaCl 2·6H 2 O或0.87g CaCl 2·sub。 2 H sub 2 O
      用蒸馏的H 2 O调至1L,将pH调节至6.9,并通过高压灭菌30分钟灭菌
    2. Fahraeus培养基(Vincent,1970)
      0.132g/L CaCl 2
      0.120g/L MgSO 4。 7H 2 O 0.100g/L KH 2 PO 4 sub/
      2H 2 O O 5 mg/L Fe-柠檬酸盐 各自为0.07mg/L的MnCl 2·2 H 2 O·CuSO 4·sup。 ZnCl 2,H 3 BO 3和Na 2 SO 4,其中H 2 O 3,H 2 O 3, /sub>MoO4.2 H sub 2 O




    1. De Oliveira,L。和Graham,P。(1990)。 根瘤菌(Rhizobium leguminosarum)菌株之间的结瘤和竞争能力速度 。 Arch Microbiol 153(4):311-315。
    2. Bhuvaneswari,T.V.,Turgeon,B.G。和Bauer,W.D。(1980)。 由大豆根瘤菌感染大豆(Glycine max L.Merr)的早期事件:I。Localization of infectible root cells. Plant Physiol 66(6):1027-1031。
    3. Toro,A。(1996)。 根瘤菌 - 共生共生中的结瘤竞争力。 < em J World J Microbiol Biotechnol。 12(2):157-162。
    4. Triplett,E.W。和Sadowsky,M.J。(1992)。 豆科植物结瘤竞争遗传学。 Annual Reviews in Microbiology 46(1):399-422。
    5. Vincent,J.M。(1970)。 根瘤菌的实际研究手册 根瘤细菌的实际研究手册。
    6. Weir,B.S。(2012)。 根瘤菌目前的分类法 NZ Rhizobia网站。
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    引用:Nelson, M. S., Chun, C. L. and Sadowksy, M. J. (2015). Evaluation of Nodulation Speed by Sinorhizobium Strains. Bio-protocol 5(15): e1554. DOI: 10.21769/BioProtoc.1554.