Rhizobium isolation and Trifolium inoculation

Presented by Julia Coffey

IntroductionThis 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.

BackgroundNitrogen 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 O2

• Plant maintains low oxygen environment• Leghaemoglobin- high affinity for O2

– 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

Stages of Nodule formation

Preinfection- Bacteria is attracted to roots by plant exudates and negative charge in rhizosphere.

Infection

Nodule formation

Nodule function

Infection process

Root-hair adhesion (lectin binding)

Root-hair curling Root-hair penetration Bacterial thread formationCell to cell proliferation of

infection thread and bacterial division

Cortical cell proliferation Bacteroid formation

NodulationCell to cell infection spreads by invasive filaments

or intercellular passagesVascular tissue surrounds nodule Leghaemoglobin productionNitrogen fixation via nitrogenase enzyme

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

Collection of nodulesAll 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 H2O2 for 4 minutes.

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.

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.

Seed sterilizationEach 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.

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.

Growth of nodules in vitroThe 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.

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.

ResultsEach group entered data into an excel spreadsheet

including:Inoculant sourceTotal number of nodulesNumber of effective nodulesDays since germinationRoot lengthPlant height

The experiment showed only the clover and lupine inoculants to induce nodulation in clover seedlings. Only the clover inoculant produced active nodules.

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.

DiscussionThe 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.

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

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,