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Rhizobium isolation and
Trifolium inoculation
Presented by Julia Coffey
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 freeliving.
Rhizobial bacteria are symbionts to 600 Genera and
1800 spp. of Leguminaseae.
Many studies on Trifolium spp.
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 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 formation
Cell to cell proliferation of
infection thread and bacterial
division
Cortical cell proliferation
Bacteroid formation
Nodulation
Cell to cell infection spreads by invasive filaments
or intercellular passages
Vascular tissue surrounds nodule
Leghaemoglobin production
Nitrogen 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 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 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 yeastmannitol broth in an Erlenmeyer
flask.
The flasks were secured in a shaker
bath at 26 °C overnight.
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.
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 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.
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.
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.
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.
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.
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,