SECTION D
INOCULATION TECHNOLOGY INTRODUCTION
The agricultural benefits possible from use of selected,
high-nitrogen fixing strains of rhizobia can be realized only
when farmers obtain and properly use high-quality inoculants on
their legume seeds or soil before planting.
Technology on
growing rhizobia, preparing inoculants with suitable carrier
materials, and distributing viable inoculants to farmers is
essential.
This section is concerned with inoculant production
and use.
Culture of Rhizobia
Rhizobia are easy to grow in the laboratory.
aerobic and also microaerophilic.
These bacteria are
They require aeration which
may be provided by using a mechanical shaker or by bubbling
sterile air through the medium.
Rhizobia grow best at 25-30C.
The medium must supply energy, a source of nitrogen, certain
mineral salts, and growth-factors.
Most commonly used is a yeast
extract mannitol mineral salts medium, but if cost or
availability is a concern, sucrose or glycerol may be
substituted.
Rhizobia grows best at 25o to 30oC.
Vessels or fermenters vary
in size from a few milliliters up to several thousand liters.
Incubation time required will vary with size of seed or starter
inoculum.
Large inocula decrease the incubation time needed to
attain the 5 X 108 to 1 X 109 rhizobial cells per milliliter
considered necessary, particularly when using a non-sterile
carrier material.
The broth culture should be checked frequently for purity and any
abrupt change in pH which indicates contamination.
Prior to
incorporation with the carrier material (peat, coal, etc.) the
culture should be checked serologically (agglutination, FA etc.)
with its homologous antiserum.
Incorporation into Carrier
The properties of a good carrier material are: No toxicity to
rhizobia, good moisture absorption capacity (for example not more
than 15% moisture in most peats prior to addition of culture),
suitable pH (6.5-7.0), fine particle size for better adherence to
seed (70 to 100% through 200 mesh screen), free of lump-forming
materials, and in ample quantities at moderate cost.
Non-availability of peat in some countries has prompted trial of
a wide range of substitutes, e.g., coal, charcoal, bagasse,
filter-mud, ground plant residues, and combinations of these with
soils.
None has proven as consistent in its ability to afford
adequate survival of rhizobia as peat.
Extraction, drying, and milling of peat are the most capital
intensive aspects of inoculant production.
The dried peat is
milled to 100-200 mesh and neutralized to pH 6.5-7.0, preferably,
with precipitated calcium carbonate.
Both sterilized and non-
sterilized peats are used in commercial production systems.
Sterile peat (gamma-irradiated at 2.5 – 5.0 megarads or
autoclaved) is generally accredited with better rhizobia survival
characteristics than non-sterile peat.
Heat sterilization of
some peats has been found to produce undesirable changes and to
release toxins.
Sterilization by gamma radiation is preferred.
Under commercial conditions in the United States, quality-tested
broth cultures are incorporated, one liter per kilogram of peat,
and packaged in thin gauge (0.05 mm) polyethylene bags.
Bags of
this specification permit gas exchange while minimizing moisture
loss from the inoculant.
Inoculants are also produced by aseptic
injection of quality tested broth cultures in packages of
presterilized peat.
Inoculants are matured for about 2 weeks at 25-30C to attain
maximum numbers of around 109 - 1010 cells/g of inoculant.
Thereafter, most inoculants are maintained under refrigeration
(4oC). Some inoculants have better survival at 26o – 28oC.
The
final moisture content of the peat inoculant should be 40-60% on
a wet weight basis for inoculants produced with presterilized
peat.
A lower moisture content (30-40%) is preferred for better
rhizobia survival in non-sterile peat.
Batches of inoculants are usually sampled for a check on their
quality at the time of leaving the production plant.
This is
done by the direct plate count in the case of inoculants based on
sterile carriers.
When non-sterile carrier material is used,
inoculant quality can only be tested adequately by a plant
infection test which lasts for about three weeks and which
conflicts with a need to distribute the inoculant quickly.
Inoculants must bear an expiration date and have an absolute
minimum of 106 viable rhizobia/g and 108 viable rhizobia/g for
non-sterile and presterilized peat, respectively, at that time.
In the United States credit for returned inoculants that have
passed their expiration date is an essential facet of inoculation
technology acceptance by farmers and is the standard policy of
the more reputable inoculant producers.
Soil and Seed Inoculation
The essence of legume inoculation is the placement of such a
large population of a highly effective nitrogen-fixing rhizobia
that is compatible with the host legume variety in close
proximity to the emerging radicle that the majority of the
nodules which form contain the introduced rhizobia.
It is important to assess the need to inoculate a particular
legume at a specific site.
Sometimes an adequate population of
effective native strains will ensure ample nodulation without
inoculation.
Alternatively, an inoculant strain may not survive
in adequate numbers or be sufficiently competitive against the
native rhizobia, and yield benefits are unlikely from
inoculation.
The most common means of introducing rhizobia to the soil is as
seed-applied inoculant.
In its simplest (and least satisfactory)
form, peat inoculant is mixed with water to form a slurry and
mixed with the seeds.
Better results are obtained when the
inoculant is coated on the seed with an adhesive.
An adhesive
increases the amount of inoculant that will adhere to the seed.
A good inoculant adhesive must be nontoxic to the rhizobia and
provide protection during planting and in the soil.
Gum Arabic
has these properties, but is expensive to farmers and not readily
available at many locations.
Other adhesives used successfully
include methyl ethyl cellulose, sucrose solutions, and vegetable
oils.
An additional coating of calcium carbonate, rock phosphate
or other pelleting material can enhance the success of
inoculation.
This is often done when adverse weather conditions
prevent immediate sowing of inoculated seeds, as protection
against insects in the soil, when the soil is hot and dry or very
acidic, or as protection against pesticides.