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Quantitative Evaluation of Competitive Nodulation among Different Sinorhizobium Strains
不同中华根瘤菌菌株中竞争性结瘤的量化评估   

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Abstract

Legumes play a vital role in global food supply because they are uniquely capable of fixing atmospheric nitrogen (N) through symbioses with root and stem nodule bacteria, collectively called the rhizobia. These 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). The symbiotic interaction is important for agricultural productivity, especially in less developed countries where nitrogen fertilizer is expensive. However, nodulation ability and competitiveness have practical importance in agricultural production, because the inoculation of efficient rhizobia is often unsuccessful, due to large part to the presence of competitive populations of ineffective indigenous rhizobia in soils (Toro, 1996; Triplett and Sadowsky, 1992). This protocol allows one us to quantitatively evaluate the relative nodulation competitiveness of Sinorhizobium strains.

Materials and Reagents

  1. Two Sinorhizobium meliloti (S. medicae) strains (strain A and B) with different intrinsic antibiotic resistance (e.g. Strain A is neomycin sensitive and strain B is neomycin resistant at a certain concentration of neomycin)
    Note: Alternately, strains can be differentiated by using gfp and rfp, antisera, or other marker genes (Triplett and Sadowsky, 1992).
  2. Medicago truncatula seeds
  3. Sodium hypochlorite
  4. Sodium chloride (Macron Chemical, catalog number: 7581-06 )
  5. Concentrated sulfuric acid (Thermo Fisher Scientific, catalog number: A300-212 )
  6. Ethanol (Decon Labs, catalog number: 2701 )
  7. Antibiotics
  8. Sunshine mix #5:Turface mixture at 1: 1 ratio (SunGro Horticulture)
  9. Turface MVP (Profile Product LLC)
  10. Tryptone (BD, catalog number: 211699 )
  11. Yeast extract (Thermo Fisher Scientific, catalog number: DF0127071 )
  12. CaCl2.2H2O (Thermo Fisher Scientific, catalog number: C79-3 )
  13. Agar (Sigma-Aldrich, catalog number: A1296 )
  14. KNO3 (Thermo Fisher Scientific, catalog number: P263-500 )
  15. Ca(NO3)2.4H2O (Acros, catalog number: 423535000 )
  16. Ca(H2PO4)2 (Spectrum, catalog number: C1145 )
  17. MgSO4.7H2O (J.T.Baker®, catalog number: 2504 )
  18. Fe-EDTA (Sigma-Aldrich, catalog number: E6760-5000 )
  19. MnCl2 (Thermo Fisher Scientific, catalog number: M33-500 )
  20. H3BO3 (Mallinckrodt, catalog number: 2549 )
  21. ZnSO4.7H2O (J.T.Baker®, catalog number: 4382 )
  22. NaMoO4 (Sigma-Aldrich, catalog number: S6646 )
  23. CuSO4.5H2O (Thermo Fisher Scientific, catalog number: C-493 )
  24. K2SO4 (EMD Millipore, catalog number: PX1595-1 )
  25. CaSO4.2H2O (Sigma-Aldrich, catalog number: C3771 )
  26. Paraffin (Thermo Fisher Scientific, catalog number: P-21 hard )
  27. TY medium (see Recipes)
  28. N2-free nutrient solution (see Recipes)
  29. Sterile paraffin coated sands (see Recipes)

Equipment

  1. MAGENTA® vessel GA-7 (Sigma-Aldrich, catalog number: V8505 ) and cotton rope (diameter ¼ inch) for Leonard jar assemblies
  2. Filter paper P5 (Thermo Fisher Scientific, catalog number: 09-801C )
  3. Plant growth chamber (TC2, Environmental Growth Chambers as an example but other plant growth chambers can be used)
  4. Autoclave

Procedure

  1. Medicago seed sterilization
    1. Place seeds in 15 ml plastic falcon tube. Add 3 ml of sulfuric acid to expose the seed for 5-8 min with occasional mixing in a fume hood with personal protective equipment.
    2. Rapidly remove the acid with pipet, and wash 4 times with sterilized double-distilled water.
    3. Soak the seeds in 10% sodium hypochlorite for 90 sec.
    4. Rinse 8 times with sterile water.
    5. Place the seeds in a petri dish on a sterile paper filer wetted with 2 ml sterile water.
    6. Place seeds in the refrigerator at 4 °C (in the dark) for 3-5 days.
    7. Move seeds to room temperature (in the dark) and let sit for 1 day to pre-germinate.

  2. Sinorhizobium strains
    1. Sinorhizobium meliloti strains are grown in TY medium for 2 days, with shaking.
    2. The cultures are centrifuged at 8,000 x g for 10 min and washed twice with sterile 0.85% sodium chloride.
    3. The resultant cultures are diluted to107 cells/ml with sterile 0.85% sodium chloride.
    4. Prepare inoculant mixture using the diluted 107 cells/ml culture. The proportion of two strains can vary from 1:1,000 to 1,000:1.

  3. Co-inoculation and plant growth condition
    1. Prepare sterile Leonard jar assemblies containing mixture of Sunshine mix #5 and Turface MVP at 1:1 ratio. Nitrogen-free plant nutrient solution was prepared for watering.
    2. Plant three germinated seeds in 1 cm deep hole and lightly cover the seeds with soil in each Leonard jar.
    3. Inoculate each seed with 1 ml of inoculant mixtures using a pipette, where the proportion of two strains varied from 1:1,000 to 1,000:1. We suggest using 5 different proportional inoculant mixtures, uninoculated controls, and control plants inoculated with a single strain.
    4. The jars are incubated in a plant growth chamber at 25 °C with a 16-h light condition and 21 °C for 8 h in the dark. Humidity should be between 50-70%. Light conditions should be 200-350 µmol/m/s.
    5. After one week, cover soil with sterilized paraffin coated sands and remove extra plants by cutting with a sterile forceps. Each pot only contains 1 plant.

  4. Recovery and identification of Sinorhizobium strains in nodules
    1. After 28 days of cultivation, 10-15 nodules are randomly collected from each plant root, and placed into 1.5 ml centrifuge tube by cutting the root about 0.5 cm on each side of the nodule.
    2. The surface of the nodules are sterilized by immersion in 95% ethanol for 10 sec and then in 2% (v/v) solution of sodium hypochlorite for 5 min.
    3. The nodules are rinsed with sterile water, 5 times, and transferred into a 96-well microtiter plate containing 100 μl of sterile 0.85% NaCl.
    4. The surface-sterilized nodule in each well is crushed by using a sterile toothpick.
    5. The suspension is streaked on TY plates.
    6. Three single colonies from each TY plate were transferred onto a TY plates containing antibiotic at specific concentrations that each strain is resistant to.
    7. Strains in nodules are determined by the antibiotic resistance.

  5. Quantitative evaluation of competitive nodulation
    1. The competitive ability for nodulation is quantified by nodule occupancy rates in seven inoculant mixtures, with the proposition of two strains ranging from 1:1,000 to 1,000:1.
    2. Nodule occupancy rate of strain A is calculated by dividing the number of nodules containing strain A (NA) by the total nodules (NA+ NB) tested.
    3. A strain which has statistically greater nodule occupancy rate when two strains co-inoculated at a 1:1 ratio (determined by microscopic count) has greater competitive ability for nodulation over the other strain.
    4. The competitiveness is quantitatively evaluated by the linear relationship between the logarithm of ratio of the numbers of nodules formed by each of two strains (NA/NB) and the logarithm of ratio of the cell numbers of two strains (IA/IB): log (NA/NB) = logCAB + k•log (IA/IB) (Amarger and Lobreau, 1982).
    5. The intercept of the regression line, the CAB value is an index representing the competitiveness of strain A in relation to that of strain B. CAB is close to 1 when two strains have equal competitiveness for nodulation. If CAB is greater than 1, stain A has greater nodulation competitiveness in relation to strain B. As an example, the nodulation competition between S. medicae WSM419 wild-type and its isogenic nolR mutant was presented in a paper by Sugawara and Sadowsky (2014).

Representative data



Figure 1. Experimental protocol for recovery and identification of Sinorhizobium meliloti strains in nodules of the host legume Medicago truncatula

Recipes

  1. TY medium
    5 g tryptone
    3 g yeast extract
    1.3 g CaCl2.6H2O or 0.87g CaCl2.2H2O
    Bring it to 1 L with distilled H2O and sterilize by autoclaving
    For the plate, 1.5% agar is added
  2. N2-free nutrient solution (Bucciarelli et al., 2006)
    KNO3
    15 mM
    Ca(NO3)2.4H2O
    12.5 mM
    Ca(H2PO4)2
    1 mM
    MgSO4.7H2O
    1 mM
    Fe-EDTA
    0.01 mM
    MnCl2
    0.004 mM
    H3BO3
    0.02 mM
    ZnSO4.7H2O
    0.0004 mM
    NaMoO4
    0.0001 mM
    CuSO4.5H2O
    0.0001 mM
    K2SO4
    12.5 mM
    CaSO4.2H2O
    9 mM
  3. Sand
    1. Dissolve 1 g of paraffin into 100 ml chloroform (mix in the hood, takes time to dissolve).
    2. Pour dissolved paraffin on 2 kg white fine sand in a metal pan.
    3. Mix paraffin/chloroform thoroughly with sand and evaporate the chloroform overnight inside hood.
    4. Distribute paraffin coated sand into an autoclavable container and autoclave for one hour.

Acknowledgments

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

References

  1. Amarger, N. and Lobreau, J. P. (1982). Quantitative study of nodulation competitiveness in Rhizobium strains. Appl Environ Microbiol 44(3): 583-588.
  2. Bucciarelli, B., Hanan, J., Palmquist, D. and Vance, C. P. (2006). A standardized method for analysis of Medicago truncatula phenotypic development. Plant Physiol 142(1): 207-219.
  3. Sugawara, M. and Sadowsky, M. J. (2014). Enhanced nodulation and nodule development by nolR mutants of Sinorhizobium medicae on specific Medicago host genotypes. Mol Plant Microbe Interact 27(4): 328-335.
  4. Toro, A. (1996). Nodulation competitiveness in the Rhizobium-legume symbiosis. World J Microbiol Biotechnol 12(2): 157-162.
  5. Triplett, E. W. and Sadowsky, M. J. (1992). Genetics of competition for nodulation of legumes. Annu Rev Microbiol 46: 399-428.
  6. Weir, B. S. (2012). The current taxonomy of rhizobia. NZ Rhizobia website.

简介

豆类在全球食物供应中起着至关重要的作用,因为它们独特地能够通过与根和茎结节细菌的共生固定大气氮(N),统称为根瘤菌。这些通常包括根瘤菌属,中生根瘤菌属,中华根瘤菌属( Ensifer )和 Bradyrhizobium 属中的细菌,的细菌现已显示与几种豆科物种形成根瘤共生(Weir,2012)。共生相互作用对于农业生产力是重要的,特别是在其中氮肥昂贵的欠发达国家中。然而,结瘤能力和竞争力在农业生产中具有实际重要性,因为高效根瘤菌的接种通常不成功,这主要是由于土壤中存在无效的土着根瘤菌的竞争性群体(Toro,1996; Triplett和Sadowsky, 。该协议允许我们定量评价中华根瘤菌菌株的相对结瘤竞争性。

材料和试剂

  1. 具有不同固有抗生素抗性的两种中华根瘤菌(EM)中华根瘤菌(菌株A和B)(例如菌株A是新霉素敏感和菌株 B是在某种浓度的新霉素抗新霉素) 注意:或者,可以通过使用gfp和rfp,抗血清或其他标记基因来区分菌株(Triplett和Sadowsky,1992)。
  2. Medic属truncatula 种子
  3. 次氯酸钠
  4. 氯化钠(Macron Chemical,目录号:7581-06)
  5. 浓硫酸(Thermo Fisher Scientific,目录号:A300-212)
  6. 乙醇(Decon Labs,目录号:2701)
  7. 抗生素
  8. 阳光混合物#5:比例为1:1的Turface混合物(SunGro Horticulture)
  9. Turface MVP(简介产品有限责任公司)
  10. 胰蛋白胨(BD,目录号:211699)
  11. 酵母提取物(Thermo Fisher Scientific,目录号:DF0127071)
  12. (赛默飞世尔科技公司,目录号:C79-3)。
  13. 琼脂(Sigma-Aldrich,目录号:A1296)
  14. KNO 3(Thermo Fisher Scientific,目录号:P263-500)
  15. (Acros,目录号:423535000)。
  16. (光谱,目录号:C1145)和/或
  17. MgSO 4·7H 2 O(J.T.Baker ,目录号:2504)
  18. Fe-EDTA(Sigma-Aldrich,目录号:E6760-5000)
  19. MnCl 2(Thermo Fisher Scientific,目录号:M33-500)

  20. (Mallinckrodt,目录号:2549)

  21. O(J.T.Baker ,目录号:4382)
  22. NaMoO 4(Sigma-Aldrich,目录号:S6646)
  23. (Thermo Fisher Scientific,目录号:C-493)

  24. (EMD Millipore,目录号:PX1595-1)
  25. (Sigma-Aldrich,目录号:C3771)
  26. 石蜡(Thermo Fisher Scientific,目录号:P-21硬)
  27. TY介质(参见配方)
  28. N 2无营养素溶液(参见配方)
  29. 无菌石蜡包被砂(见配方)

设备

  1. 用于Leonard罐组件的MAGENTA容器GA-7(Sigma-Aldrich,目录号:V8505)和棉绳(直径为1/4英寸)
  2. 滤纸P5(Thermo Fisher Scientific,目录号:09-801C)
  3. 植物生长室(TC2,环境生长室作为实例,但可以使用其他植物生长室)
  4. 高压灭菌器

程序

  1. 苜蓿种子灭菌
    1. 放置种子在15毫升塑料猎鹰管。 加入3ml硫酸 将种子暴露5-8分钟,在通风橱中偶尔混合 个人保护设备。
    2. 用移液管快速除去酸,用无菌双蒸水洗涤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上,用2ml无菌水润湿
    6. 将种子在冰箱中在4℃(在黑暗中)3-5天。
    7. 将种子移至室温(在黑暗中),让其静置1天以预发芽

  2. 中华根瘤菌菌株
    1. 将中华根瘤菌(Sinorhizobium meliloti)菌株在TY培养基中振荡生长2天。
    2. 将培养物以8,000×g离心10分钟,并用无菌的0.85%氯化钠洗涤两次。
    3. 将所得培养物用无菌0.85%氯化钠稀释至10 7细胞/ml
    4. 使用稀释的10 7个细胞/ml培养物制备接种物混合物。 两种菌株的比例可以在1:1,000至1,000:1之间变化

  3. 共接种和植物生长条件
    1. 准备含有Sunshine混合物的无菌Leonard罐组件 混合物#5和Turface MVP以1:1的比例混合。 无氮植物营养 溶液用于浇水。
    2. 在1cm深孔中种植三个发芽的种子,并在每个Leonard瓶中用土壤轻轻地覆盖种子。
    3. 使用移液管接种1毫升接种混合物的每个种子,   其中两种菌株的比例从1:1,000至1,000:1变化。 我们 建议使用5种不同比例接种混合物,未接种 对照和用单一菌株接种的对照植物。
    4. 将瓶在25℃的植物生长室中孵育16小时 光照条件和21℃在黑暗中8小时。 湿度应该 在50-70%之间。 光照条件应为200-350μmol/m/s
    5. 一周后,用灭菌的石蜡包被的砂覆盖土壤 通过用无菌镊子切割除去额外的植物。 每罐只 包含1株植物。

  4. 恢复和鉴定结节中的中华根瘤菌菌株
    1. 培养28天后,从中随机收集10-15个结节 每个植物根部,并置于1.5ml离心管中通过切片 根结节每侧约0.5厘米
    2. 表面的 结节通过浸入95%乙醇中10秒然后消毒 在2%(v/v)次氯酸钠溶液中5分钟
    3. 的 结节用无菌水冲洗5次,并转移至 含有100μl无菌0.85%NaCl的96孔微量滴定板。
    4. 使用无菌牙签将每个孔中的表面消毒的结节压碎
    5. 将悬浮液在TY板上划线
    6. 将来自每个TY板的三个单菌落转移到TY 板含有在每种菌株的特定浓度的抗生素   耐。
    7. 结节中的菌株由抗生素抗性决定。

  5. 竞争性结瘤的定量评价
    1. 结节的竞争能力由结节占据量化   率在七个接种混合物,与两个菌株的命题 范围为1:1,000至1,000:1。
    2. 结节占位率   A通过除含有菌株A的结节数来计算 (N A A + N B)的总结节(N A A)。
    3. 一种菌株 两种菌株统计学上更大的结节占据率 以1:1的比例(通过显微计数确定)共接种 比其他菌株更高的结瘤竞争能力。
    4. 竞争力由线性定量评估 结节数之比的对数之间的关系 由两个菌株(N A A/N B B)中的每一个形成的比率的对数和 两个菌株的细胞数目(I A A/I B):对数(N A A/N B B) logC AB + k·log (I /I )(Amarger和Lobreau,1982)。
    5. 截距 回归线,C AB值是表示 菌株A的相对于菌株B的竞争性.C AB 接近1,当两个菌株具有同等的竞争性结瘤。 如果C sub AB大于1,则染色A具有更高的结瘤竞争性  关于菌株B.作为例子,结瘤竞争 之间。药物 WSM419野生型及其同基因nolR 突变体 在Sugawara和Sadowsky的论文(2014)中提出。

代表数据



图1. 恢复和鉴定 中华根瘤菌 宿主豆结核中的菌株的实验方案 Medicago truncatula

食谱

  1. TY介质
    5克胰蛋白酶
    3g酵母提取物
    1.3g CaCl 2·6H 2 O·6H 2 O或0.87g CaCl 2·2H 2 O.H 2 O 2 O
    用蒸馏的H 2 O将其调至1L,并通过高压灭菌
    灭菌 对于板,加入1.5%琼脂
  2. N无营养物溶液(Bucciarelli等人,2006)
    KNO 3
    15 mM
    Ca(NO 3)2 sub 2 4H 2 O
    12.5 mM
    Ca(H 2 PO 4)sub 2 2/2
    1 mM
    MgSO 4。 。 O
    1 mM
    Fe-EDTA
    0.01 mM
    MnCl 2
    0.004 mM
    H 3 BO 3
    0.02 mM
    ZnSO 4 。 7H O
    0.0004 mM
    NaMoO 4
    0.0001 mM
    CuSO 4 5H sub 2 O
    0.0001 mM
    K 2 SO 4
    12.5 mM
    CaSO 4 2H O
    9 mM

    1. 将1g石蜡溶解在100ml氯仿中(在通风橱中混合,需要时间溶解)。
    2. 将溶解的石蜡倒在金属盘中的2kg白色细砂上。
    3. 将石蜡/氯仿用砂彻底混合,并在罩内蒸发氯仿过夜。
    4. 将石蜡包被的砂子分配到高压灭菌容器中并高压灭菌1小时。

致谢

本研究得到国家科学基金资助1237993。

参考文献

  1. Amarger,N。和Lobreau,J.P。(1982)。 根瘤菌属菌株的结瘤竞争力的定量研究。 em> Appl Environ Microbiol 44(3):583-588。
  2. Bucciarelli,B.,Hanan,J.,Palmquist,D.and Vance,C.P。(2006)。 分析Medic藜表皮发育的标准方法。 Plant Physiol 142(1):207-219。
  3. Sugawara,M。和Sadowsky,M.J。(2014)。 通过norR突变体增强中华根瘤菌药物的结瘤和结节发育, > Medicago 宿主基因型。 Mol Plant Microbe Interact 27(4):328-335。
  4. 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。
  5. Triplett,E.W。和Sadowsky,M.J。(1992)。 豆科植物结瘤竞争遗传学。 Annu Rev Microbiol 46:399-428。
  6. Weir,B.S。(2012)。 根瘤菌目前的分类法。新西兰根瘤菌网站。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chun, C. L., Nelson, M. S. and Sadowksy, M. J. (2015). Quantitative Evaluation of Competitive Nodulation among Different Sinorhizobium Strains. Bio-protocol 5(15): e1555. DOI: 10.21769/BioProtoc.1555.
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