1. Rhizobium isolation and Trifolium inoculation Presented by Julia Coffey

2. Introduction  This laboratory was conducted to determine specificity in the nodulating symbiosis of clover (Trifolium sp.) and Rhizobia spp..  Nodulation is a symbiosis between N-fixing prokaryotes and plant roots.  The relationship is facultative: both can exist free- living.  Rhizobial bacteria are symbionts to 600 Genera and 1800 spp. of Leguminaseae.  Many studies on Trifolium spp.

3. Background  Nitrogen Fixation requirements: –pH- Rhizobium can not survive in pH<4.3 –Carbon- Rhizobium exist free living as aerobic heterotrophs –Oxygen status- Nitrogenase enzyme is sensitive to O 2 Plant maintains low oxygen environment Leghaemoglobin- high affinity for O 2 –Protein from plant –Haeme from bacteria –Supply of Nitrogen- Nitrogen fixation occurs only when it is needed due to high carbon demand.  “Two organisms forming a specially functioning organ together” Erik Thuesen

5. Stages of Nodule formation  Preinfection- Bacteria is attracted to roots by plant exudates and negative charge in rhizosphere.  Infection  Nodule formation  Nodule function

6. Infection process  Root-hair adhesion (lectin binding)  Root-hair curling  Root-hair penetration  Bacterial thread formation  Cell to cell proliferation of infection thread and bacterial division  Cortical cell proliferation  Bacteroid formation

7. Nodulation  Cell to cell infection spreads by invasive filaments or intercellular passages  Vascular tissue surrounds nodule  Leghaemoglobin production  Nitrogen fixation via nitrogenase enzyme

8. Materials and Methods The laboratory consisted of a multiple step procedure:  Collection of nodules and isolation of bacteria  Secondary isolation and pure culture of bacteria  Inoculation of clover seeds with bacteria and growth of nodules in vitro  Determination of successful nodulation and specificity

9. Collection of nodules  All equipment was washed in water, rinsed in ethanol, and de-ionized water and flamed for sterility.  Nodulating roots of alder, clover, scotch broom, and lupine were obtained from TESC campus area.  The nodules were cut from the roots and thoroughly washed with water and a touch of detergent, rinsed in ethanol, and immersed in H 2 O 2 for 4 minutes.

10. Isolation of Bacteria  After soaking the nodules individually, they were crushed in a glass Petri dish. Using an inoculating loop, the milky fluid from each nodule was used to inoculate three agar plates.  The plates were labeled and placed in an incubator at 26°C.

11. Secondary isolation and pure culture of bacteria  After 4 days of incubation, an isolated, uniform colony was selected from one of the plates for each source of inoculant.  Using an inoculating loop, the colony was transferred to a yeast- mannitol broth in an Erlenmeyer flask.  The flasks were secured in a shaker bath at 26 °C overnight.

12. Seed sterilization  Each group soaked ~25 seeds in ethyl alcohol for 5 minutes and commercial bleach for 20 minutes.  Seeds were aseptically transferred to sterile plastic Petri dish and rinsed 5 times in sterile DI water.  6 seeds were transferred to each of 4 sterile Petri dishes, 3 with inoculant solution and 1 control.

13. Inoculation of Clover Seeds  12 N-free agar seedling slants were prepared with enough DI water to over ~50% of the slant.  Using measured pipettes 100μl of each yeast-mannitol inoculant was transferred to three slants.  Two seeds were placed in each slant with inoculant. Three control slants were also planted.

14. Growth of nodules in vitro The slants were wrapped and capped in foil to protect the seeds from light and contamination. The slants were incubated in a growth chamber on a 12 hour light/dark cycle. Each of the nine groups monitored their seedlings on an average of 2-3 days and watering as needed.      

15. Data collection  After 24 days in the growth chamber, the seedlings were removed from the slants.  The nodules were counted, and dissected to determine activity by the presence of leghaemoglobin.  The growth of root and shoot were measured for each plant.

16. Results  Each group entered data into an excel spreadsheet including:  Inoculant source  Total number of nodules  Number of effective nodules  Days since germination  Root length  Plant height  The experiment showed only the clover and lupine inoculants to induce nodulation in clover seedlings. Only the clover inoculant produced active nodules.

17. Results  Clover- of the 29 plants, 16 had nodules, 11 of which produced leghaemoglobin indicating active nodulation. Out of 100 nodules observed 80% were active.  Lupine- of the 24 plants, 4 plants produced nodules none of which were active.  Scotch broom and Alder failed to produce any nodules.

18. Discussion  The experiment demonstrated that the specificity in this relationship is broad enough for clover plants to initiate symbiosis with Rhizobium inoculant from lupine.  The nodules of the seedlings inoculated by lupine Rhizobium were inactive, indicating that the Trifolium symbiosis is specific enough to inhibit nodule function in non-compatible strains of Rhizobium.  The specificity observed in this experiment can be attributed to molecular interactions in the determination of Rhizobial symbiosis.

19. Molecular interactions in the determination of Rhizobial symbiosis:  Flavonoid inducers- Produced by plant –Released in root hair zone specific for attracting Rhizobium –Range of favonoids produced by each plant host for specific Rhizobium species  Nod proteins- Rhizobium nod genes code for Nod proteins –NodD is an important determinate of host range –Activates transcription of Nod factors  Nodulation factors- –Different bacteria produce species specific Nod factors –Cause changes in plant root Enod proteins: –root hair curling, infection tube, symbiosome membrane etc. Late nodulins: Leghaemoglobin  Lectins- –Sugar binding proteins involved in many cell-cell interactions –Important in recognition

20. References cited: Brady and Weil (2001). The Nature and Properties of Soils 13th Edition. Douglas, A.E. (1994). Symbiotic interactions. Oxford University Press, Paracer, S. & Ahmadjian, V., (2000). Symbiosis: an introduction to biological associations. Oxford University Press,