EXERCISE 17
SELECTING EFFECTIVE STRAINS OF RHIZOBIA IN POTTED FIELD-SOIL
Strains of rhizobia previously screened in Leonard jars are
evaluated further in potted field soil.
The effectiveness of
mixed and single strain inocula are compared.
Infective native
rhizobial populations in field soil are determined.
Key steps/objectives
1)
Culture rhizobia
2)
Collect soil from test field
3)
Prepare soil, determine pH and total N content
4)
Pot the soil
5)
Determine water holding ability (field capacity) of soil
6)
Apply fertilizer
7)
Plant and inoculate surface sterilized seeds
8)
Thin seedlings to desired number
9)
Inspect for nodulation and perform MPN counts
10)
Water and observe plants
11)
Harvest plants, examine nodulation
12)
Analyze data
(a)
Designing the experiment and treatments
The experimental design is a randomized complete block with three
blocks as in Exercise 16.
There are 18 treatments: 15 inoculated
(14 single strain inoculations and one treatment receiving a
mixed broth inoculum comprising the three best strains selected
in Leonard jars from Exercise 16); a plus-N control without
inoculation; and two sets of non-inoculated controls.
At 2 weeks
the extra set of the non-inoculated controls is removed for
inspection for nodulation by native rhizobia.
If nodulation is
observed in the non-inoculated controls, initiate MPN counts of
the native population using the soil set aside for this purpose.
Pots are sown with eight seeds and four plants are maintained for
the experiment upon thinning.
(b)
Preparing the inoculum
(Key step 1)
All of the 14 cultures of B. japonicum used in Exercise 16 are
evaluated in soil.
Inoculate each strain into 70 ml of
yeast-mannitol broth contained in 125 ml Erlenmeyer flasks.
Allow strains to grow for 5-7 days to reach maximum turbidity
(approximately 1 x 109 cells ml-1).
To prepare the mixed
inoculum, pipette 10 ml of the fully grown broth culture of each
of the three best strains into a clean 125 ml Erlenmeyer flask.
Use a fresh pipette for each strain.
Mix the contents thoroughly
by swirling.
(c)
Choosing the site for collecting soil
The ideal site for soil collection is the one where the field
experiment (which follows the pot experiment) is to be
conducted.
The site soil should be low in nitrogen.
The native
rhizobial population should be less than 103 rhizobia per g soil;
no previous history of inoculation and cultivation with the
intended legume; no water-logging or salinity problems.
In practice, these prerequisites may not be met in the chosen
site.
This, however, should not deter experimentation with a
particular soil.
(d)
Collecting, preparing, and potting field soil
(Key steps 2, 3, and 4)
With a steel spade or other suitable implement, obtain field soil
from a depth of 10-15 cm.
within a soil type.
Soil samples should be taken randomly
Collect and transport the soil
(approximately 150 kg) in strong plastic bags to a clean room.
Spread large pieces of clean cardboard on the floor and cover
with thick, clean, plastic sheets or tarpaulins.
Empty the bags
of soil onto the plastic to pool all the collected soil.
the soil and allow it to air dry.
Mix the soil thoroughly and
remove debris (e.g., stones, roots, leaves, etc.)
with a wooden mallet.
Spread
Break lumps
Sift the soil using a 5 mm mesh screen.
Take a sample to determine the soil pH using a pH-meter.
soil is acid, add lime to bring the pH to 6.0-6.5.
If the
Mix the soil
and lime thoroughly and allow to equilibrate for at least 7
days.
During the equilibration period, cover the soil with a
plastic sheet.
Use one of the methods shown in Appendix 16 to
calculate the amount of lime needed to adjust the pH level of the
soil.
Obtain strong PVC (polyvinylchloride) pots.
Pots of 15-16 cm
diameter, and 18 cm height with a capacity of just over 3 l and
with at least one hole on the bottom, are suitable for potting.
Pots should be clean.
Plastic bags of suitable size and
thickness will be used as inner liners for the pots.
Punch holes
(1 cm diameter) in the bottom of the bags to allow for drainage.
Position the bags in the pots and fold the open end of the bag
over the rim of the pot.
Pots of the recommended size will hold approximately 2.4-2.7 kg
of a soil high in organic matter.
Tropical soils with less
organic matter but occupying a similar volume will be heavier.
Weigh 2.4 kg of soil in each plastic bag and place in the pot.
(Any coarse balance is suitable for weighing the soil, as high
precision is not required.)
compact the soil.
Gently tamp the pots on the floor to
Soil in all pots must be tamped down to occupy
nearly the same volume to achieve similar bulk density.
Set aside 250 g of soil in a refrigerator (4C) for MPN count of
the native rhizobial population following the method described in
Exercise 5.
(e)
Adjusting moist field soil to field capacity
(Key step 5)
A soil moisture content at field capacity is suitable for most
plants.
Since the field capacity varies with different soils,
determine the field capacity for the soil under investigation.
At sowing and during initial phase of seed germination and
seedling establishment, the soil moisture should be maintained at
field capacity for better plant performance.
Determine the field
capacity of the moist field soil using the simple method
described in Appendix 21.
(f)
Applying fertilizer
(Key step 6)
The fertility of the soil must be adjusted to optimal levels to
obtain good growth of the plants.
treatments are recommended.
The following fertilizer
Rates per pot have been calculated
on the basis of 2.4 kg-soil per pot.
Phosphorus, P
100 kg P ha-1; applied as 500 kg ha-1 triple super-phosphate
(TSP*); 529 mg pot-1 (or 468 mg KH2PO4 pot-1).
Potassium, K
200 kg K ha-1; applied as 382 kg ha-1 KCl; 404.2 mg pot-1
(K2SO4 may also be used)
Magnesium, Mg
5 kg Mg ha-1; applied as 50 kg ha-1 MgS04.7H2O; 53.3 mg pot-1
Zinc, Zn
10 kg Zn ha-1; applied as 46.8 kg ha-1 ZnSO4.7H20; 49.5 mg
pot-1
Molybdenum, Mo
1.0 kg Mo ha-1; applied as 1.76 kg (NH4)6 Mo7024.H20 ha-1; 1.95
mg pot-1
Nitrogen, N (for N-control pots)
100 kg N ha-1; applied as 222 kg ha-1 urea, CO(NH2)2; 219 mg
pot-1 25% of N is applied at planting and the remaining 75%
at 3 weeks.
Prepare the fertilizers (except the insoluble triple
superphosphate) in the form of solutions and pipette them on to
the soil surface and allow to dry.
superphosphate.
Add the triple
Mix the soil in each pot thoroughly to ensure
uniform distribution of the nutrients (mixing is easily achieved
by removing the bag of soil from the pot and massaging).
(g)
Planting and inoculating the seeds
(Key steps 7 and 8)
At the planting rate of eight seeds per pot, a total of 24 seeds
are needed for each treatment in triplicate.
A grand total of
408 seeds are needed for all the 17 treatments.
From a batch of
seeds with good germination, select 500 seeds and surface
sterilize as in Appendix 10.
Allow the sterilized seeds to
imbibe water for 1 h.
Give the seeds a final rinse and plant the
seeds at a depth of 2 cm.
Inoculate each seed with 1 ml of the
culture, following the method described in Chapter 19.
Label the
treatments and assign block numbers.
Water the soil in the pots to field capacity using the data from
Step 2.
Add sterilized coarse sand mulch to control
contamination.
Randomize the pots on the greenhouse bench.
When plants are 5 days old, thin to four uniform plants per pot
as described in Exercise 15.
(h)
Inspecting non-inoculated control plants for nodulation by
native rhizobia
(Key step 9)
When plants are 3 weeks old, remove the extra set of
non-inoculated controls to inspect for nodulation by native
rhizobia.
Carefully remove the plastic bag containing the plants
from the pot and place it in a shallow basin.
open.
Slit the bag
With a gentle stream of water wash the roots.
nodulation.
Examine for
Similarly observe the remaining two pots set up for
inspection.
If nodules are present, make preparations for performing the MPN
count of rhizobia in the soil set aside for this purpose in (d).
The count may be done in growth-pouches or Leonard jars following
the method described in Exercise 5.
Weigh 100 g of the soil, dilute it in 900 ml of sterile water and
prepare a fourfold dilution series ranging from 41 - 410
dilution.
Inoculate each dilution in quadruplicate.
A fourfold
series gives more precision than a tenfold series, especially for
soils when populations are less than 1 x 103 rhizobia per g
soil.
Note that the starting sample has been diluted 1:10.
More
details on the method and calculations are given in Exercise 5.
(i)
Watering the pots and making periodic observation
(Key step 10)
During active growth and fixation, legumes will use a
considerable amount of water each day.
pots need to be watered regularly.
During this period, the
Water the pots more than once
each day if needed.
Weigh sample pots showing vigorously growing plants to determine
the volume of water needed to replace the water lost.
If there
are large differences in plant growth, pots should be watered to
weight on a pot by pot basis.
Measure out the required volume of
water in a measuring cylinder and pour into the pot without
excessively disturbing the soil.
Keep plants well watered and
make growth observations periodically as in Exercise 15.
(j) Harvesting the experiment
(Key steps 11 and 12)
Harvest the plants at 35 days.
and nodules for all treatments.
Exercise 15.
Determine dry weight of shoots
Analyze yield data as in
Requirements
(a)
Designing the experiment and treatments
No special requirements
(b)
Preparing the inoculum
Transfer chamber
Agar slant cultures of rhizobia
Yeast-mannitol broth
Shaker
Erlenmeyer flasks
Pipettes
(c)
Choosing the site for collecting soil
Soil analysis data
(d)
Collecting, preparing, and potting field soil
Steel spade
Strong plastic bags, plastic sheets, cardboard
Wooden mallet
1 cm mesh screen
pH meter
Lime (CaCO3)
PVC pots and plastic bags (inner liners for pots)
Balance for weighing potted soil
(e)
Adjusting moist field soil to field capacity
Determine field capacity (Appendix 21)
(f)
Applying fertilizer
Weighing balance
Triple superphosphate, potassium chloride, zinc sulfate,
ammonium molybdate, urea, lime, magnesium sulfate, potassium
phosphate
Pipettes (1 ml and 10 ml)
(g)
Planting and inoculating seeds
Seeds, sodium hypochlorite solution (3%), sterile water
Sterile empty beakers
Sterile pipettes, forceps, marker pens
Balance, water
Scissors, alcohol lamp, matches
(h)
Inspecting non-inoculated control plants for nodulation by
native rhizobia
Tap water, scissors, shallow basin