Materials and Reagents

Sinorhizobium meliloti strains
Medicago truncatula seeds
Sodium chloride (MACRON CHEMICAL, catalog number: 7581-06 )
Sodium hypochlorite
Ethanol (Decon Labs, catalog number: 2701 )
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 )
MnCl_2^.4 H₂O (Thermo Fisher Scientific, catalog number: M33-500 )
CuSo₄^.5 H₂O (Thermo Fisher Scientific, catalog number: C-493 )
ZnCl₂ (JT Baker, catalog number: 4326-01 )
H₃BO₃ (Mallinckrodt, catalog number: 2549 )
Na₂MoO₄^.2 H₂O (Sigma-Aldrich, catalog number: S6646 )
TY medium (see Recipes)
Fahraeus medium (see Recipes)

Equipment

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

Procedure

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).
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.
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.
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

Recipes

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
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 Na₂MoO_4^.2 H₂O
Adjust pH to 7.5 before autoclaving for 1 h for 10 L

Acknowledgments

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

References

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.
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.
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. Annual Reviews in Microbiology 46(1): 399-422.
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.
Weir, B. S. (2012). The current taxonomy of rhizobia. NZ Rhizobia website.