Materials and Reagents

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).
Medicago truncatula seeds
Sodium hypochlorite
Sodium chloride (Macron Chemical, catalog number: 7581-06 )
Concentrated sulfuric acid (Thermo Fisher Scientific, catalog number: A300-212 )
Ethanol (Decon Labs, catalog number: 2701 )
Antibiotics
Sunshine mix #5:Turface mixture at 1: 1 ratio (SunGro Horticulture)
Turface MVP (Profile Product LLC)
Tryptone (BD, catalog number: 211699 )
Yeast extract (Thermo Fisher Scientific, catalog number: DF0127071 )
CaCl₂^.2H₂O (Thermo Fisher Scientific, catalog number: C79-3 )
Agar (Sigma-Aldrich, catalog number: A1296 )
KNO₃ (Thermo Fisher Scientific, catalog number: P263-500 )
Ca(NO₃)₂^.4H₂O (Acros, catalog number: 423535000 )
Ca(H₂PO₄)₂ (Spectrum, catalog number: C1145 )
MgSO₄^.7H₂O (J.T.Baker^®, catalog number: 2504 )
Fe-EDTA (Sigma-Aldrich, catalog number: E6760-5000 )
MnCl₂ (Thermo Fisher Scientific, catalog number: M33-500 )
H₃BO₃ (Mallinckrodt, catalog number: 2549 )
ZnSO₄^.7H₂O (J.T.Baker^®, catalog number: 4382 )
NaMoO₄ (Sigma-Aldrich, catalog number: S6646 )
CuSO₄^.5H₂O (Thermo Fisher Scientific, catalog number: C-493 )
K₂SO₄ (EMD Millipore, catalog number: PX1595-1 )
CaSO₄^.2H₂O (Sigma-Aldrich, catalog number: C3771 )
Paraffin (Thermo Fisher Scientific, catalog number: P-21 hard )
TY medium (see Recipes)
N₂-free nutrient solution (see Recipes)
Sterile paraffin coated sands (see Recipes)

Equipment

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

Procedure

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.
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.
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.
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.
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 (N_A) by the total nodules (N_A+ N_B) 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 (N_A/N_B) and the logarithm of ratio of the cell numbers of two strains (I_A/I_B): log (N_A/N_B) = logC_AB + k•log (I_A/I_B) (Amarger and Lobreau, 1982).
5. The intercept of the regression line, the C_AB value is an index representing the competitiveness of strain A in relation to that of strain B. C_AB is close to 1 when two strains have equal competitiveness for nodulation. If C_AB 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

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

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

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

Amarger, N. and Lobreau, J. P. (1982). Quantitative study of nodulation competitiveness in Rhizobium strains. Appl Environ Microbiol 44(3): 583-588.
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.
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.
Toro, A. (1996). Nodulation competitiveness in the Rhizobium-legume symbiosis. World J Microbiol Biotechnol 12(2): 157-162.
Triplett, E. W. and Sadowsky, M. J. (1992). Genetics of competition for nodulation of legumes. Annu Rev Microbiol 46: 399-428.
Weir, B. S. (2012). The current taxonomy of rhizobia. NZ Rhizobia website.