Note: Descriptions are shown in the official language in which they were submitted.
133S~34
RHIZOBIUM INOCULANTS
Field of the Invention
The present invention relates to inoculants for
leguminous crops in general, and relates, in particular,
to a method for creating novel Rhizobium strains which are
both biologically competitive and which have high nitrogen
fixation ability, and to methods for creating these
strains.
Background of the Invention
The phenomenon of the symbiotic relationship between
legumes and the Rhizobium bacteria which nodulate their
roots is widely known. The bacteria, living in the
nodules in the legume roots, fix nitrogen directly from
the atmospheric nitrogen thereby converting nitrogen to
biologically useful nitrogenous compounds. This process
not only provides nitrogen to the plant for protein
synthesis, but also enriches the soil in which the legume
is grown by leaving nitrogenous nutrients in the soil for
later crops. Examples of legumes which are capable of
symbiotic relationship with Rhizobium bacteria are peas,
beans, alfalfa, red clover, white clover, vetch, and
133~43~
lupines.
Since other agricuLturally important plants are
unable, either directly or indirectly, to fix nitrogen
directly from the air, and thus generally are dependent
upon nitrogenous fertilizers to introduce sufficient
nitrogen content in the soil for good yield, it is quite
common for legumes to be alternated each growing season
with non-legume crops in important agricultural areas to
facilitate economical enhancement of nitrogen content in
the soil. This practice, known as crop rotation, is wide
spread. Furthermore, some legumes, and in particular
soybeans, peas, beans, and alfalfa, are important
commercial crops in their own right and growth of these
crop plants is greatly facilitated by an ample supply of
combined or fixed nitrogen.
There are a number of species of Rhizobium bacteria
and different Rhizobium species are adapted to form
symbiotic relationships, and nodules, only in the roots of
specific legumes. For example, R. japonicum nodulates the
roots of soybeans, R. trifolii nodulates the roots of
clovers, R. meliloti grows symbiotically with alfalfa and
sweet clovers, R. leguminosarum is used with peas and
vetches, and R. phaseoli nodulates the roots of common
garden beans. Therefore, in commercial agricultural
practices, it is quite a common practice to inoculate the
soil or the seeds of the legumes with a culture of the
appropriate Rhizobium species. This inoculant is commonly
done by coating the seeds before planting, dusting planted
seeds, or by spreading the inoculant in the furrows of the
planted legume seeds.
In creating and formulating a Rhizobium inoculant, it
is desirable that the Rhizobium strain contained in the
inoculant have an enhanced ability to fix nitrogen for the
benefit of the plant and for soil conditioning.
Therefore, much research has logically been conducted on
methods for improving the nitrogen fixation of Rhizobium
_3_ 1335434
species. Most of the currently commercially available
Rhizobium inoculants include strains having high nitrogen
fixing ability selected from natural populations.
However, the ability to fix nitrogen does not ensure
the survivability of the bacteria in field conditions.
Because legumes have been cultivated for many generations
in most of the important agricultural areas of the world,
many strains of Rhizobium bacteria now freely live
indigenously in important agricultural areas. The
bacteria may not have been naturally indigenous to all
these areas, but introduced strains have populated many of
these areas and since they survive freely they may be
considered effectively indigenous.
All free-living bacteria, in the soil or elsewhere,
are continually subject to environmental pressures, and
naturally tend to mutate and be selected by the pressures
of the ecological niche which they occupy to be further
adapted and competitive for that niche. The indigenous
Rhizobium strains are, of course, subject to these
pressures. Therefore many of the soils in agricultural
areas now contain indigenous Rhizobium strains which have
evolved to be adapted for survival in competitive
existence in that particular soil environment.
Accordingly, even if a farmer introduces into a field a
Rhizobium inoculant strain having high nitrogen fixing
capability, there is no guarantee that the introduced
Rhizobium strain will predominate the roots of the legume
plants. The introduced inoculant strain promptly enters
into ecological competition with the indigenous strains
already present in the soil to nodulate the plants, and,
many times, the indigenous populations are an advantage
because they are previously selected by ecological
pressures for competition in precisely the environment of
that field or climatic area. Accordingly, even a high
nitrogen fixing Rhizobium strain which can effectively
nodulate the legume and fix nitrogen effectively without
-4- 1335434
competition may be an ineffective inoculant in the field
since the introduced strain may not actually populate the
roots of the legumes, but may be overwhelmed by a locally
indigenous strain, which may not have optimal nitrogen
fixation abilities. No prior teachings are known on
either the desirability of or the process for creating
novel Rhizobium strains which are both competitive in
field conditions and have high nitrogen fixation
abilities.
It has been known previously that legume root exudate
can be used to cause Rhizobium cells to develop more
lectin-binding sites. It is also known that such exudates
can be used as a pretreatment to foster and to enhance the
ability of Rhizobium to nodulate when inoculated onto a
legume plant.
Summary of the Invention
The present invention is summarized in that novel
strains of Rhizobium bacteria useful as a crop inoculant
for legume species in a selected geographic area are
created by the method of: isolating a number of Rhizobium
cultures from the geographic area; identifying the
naturally predominant Rhizobium strain in the geographic
area by comparative analysis of the cultures; exposing a
culture of the naturally predominant strain to a
mutagenic agent; and selecting among the mutagenized
bateria for mutants having high nitrogen fixation ability
in association with the legumes.
The present invention is also directed toward the
novel strains created through the use of this process.
It is an object of the present invention to provide a
method for creating novel Rhizobium strains which are both
highly naturally competitive for a selected geographic
region and also have enhanced nitrogen fixation abilities.
It is another object of the present invention to
_5_ 1335~3~
provide strains selected by this method which are more
predictably able to enhance the growth of legume crop
plants than presently available Rhizobium inoculant
strains.
The Rhizobium species created by the method in
accordance with the invention is preferably selected
from the group consisting of R. japonicum, R. trifolii,
R. lequminosarium, R. meliloti and R. phaseoli.
It is a feature of the present invention in that it is
able to select for strain competltiveness and prevalence
in particular agricultural environmental conditions
without the nece-~sity for simulating the competitive
conditions on a bacterial strain in an artifically
controlled environment, a simultation which has proven
extraordinarily difficult to perfect in practice.
To clarify the term "particular agcicultural
environmental conditions~, it has been found that geographic
regions which cover multi-state legume cultivation regions may
be determined to have sufficiently common agricultural,
enviconmental and climatic conditions that a single strain, or
at most a veey small number of strains (two or three), are
predominant in such a region. The geographic size of such a
region can be quite large as long as the same indigenous strain
or strains are predominant throughout the region. The proper
size for such a region may be determined by the extent to which
a single, or at most very few, strains are predominant over the
cegion. To be predominant over the region, the strain need not
be dominant in every field in every locale in the region, but
must be sufficiently predominant that a statistical sampling of
fields in the region will yield the predominant strain more
than any other.
_ - 5a - 133~93~
Other objects, advantages, and features of the present
invention will become apparent from the following detailed
description.
Brief Description of the Drawings
Fig. 1 is a flow chart of the methodology used to
generate high nitrogen fixing strains of the competitive
Rhizobium strains isolated and identified in accordance
with the procedure of the present invention.
Fig. 2 is a chart illustrating the enhancement of
nitrogen fixation activity obtained from one mutagenized
naturally predominant strain.
Fig. 3 i8 a chart of the enhanced nitrogen fixing
activity obtained from a second strain.
Fig. ~ is a chart illustrating enhancement of
nodulation obtained by inoculant pre-treatment.
Detailed Descripiton of the Invention
The practice of the process of creating novel
Rhizobium strains pursuant to the present invention
proceeds through a series of steps. First, it is
necessary to isolate a number of Rhizobium cultures from a
selected geographic area which are then analyzed to
identify naturally predominant strains of Rhizobium. The
identified naturally predominant strains are then subject
to mutagenesis with the mutagenized colonies being
selected for high nitrogen fixation. After high nitrogen
fixing mutants are discovered, the mutants are returned to
field test trial to determine that they both remain
competitive and remain sufficiently high nitrogen fixing
in the field to increase yield. To fully understand the
details of each of these procedures, they will each be
discussed in order.
-6- 1335434
The process of isolation of indigenous Rhizobium
cultures to begin with the identification of competitive
strains. Contrary to prior strategies for creating
Rhizobium inoculants, no effort is taXen in accordance
with the present invention to try to select for, or
engineer for, competitiveness in actual field condition.
Instead, the first step is directed toward identifying
those indigenous naturally predominant strains which are
already present in the selected geographic growing area
for the legume crop selected. There are a number of species
of Rhizobium bacteria and different Rhizobium species are
adapted to form symbiotic relationships, and nodules, only
in the roots of specific legumes. For example, R. iaponicum
nodulates the roots of soybeans, R. trifolii nodulates the
roots of clovers, R. meliloti grows symbiotically with
alfalfa and sweet clovers, R. leguminosarium is used with
peas and vetches, and R. phaseoli nodulates the roots of
common garden beans. Thus, if soybean is the legume of
interest, one would select the locales in which
soybeans are grown, and identify the strains of indigenous
Rhizobium laponicum naturally predominant in that locale.
However, any other naturally occurring Rhizobium species
described herein may be selected for use in the method of
this invention.
As used herein, the term "indigenous" does not mean
naturally occurring before the advent of mankind, since
R japonicum, for example, is an introduced species in t~e
Western ~emisphere, but refers to the strains which
naturally thrive in the soils in the agricultural areas at
present. Most soils, for example, in soybean producing
areas of the United States now have indigenous local
strains of R japonicum naturally present and the strains
have evolved to the soil types and climatic regions of the
country.
The determination and identification o~ the
competitive strains proceeds first by the large scale
collection of nodules from the legume cultivation region
_7_ 1335434
which has been selected. Legume crop fields are located
which have not been inoculated with any Rhizobium
inoculant. Plants are removed from the fields, and the
nodules are harvested from the roots of plants grown in
those fields. The nodules are surface-sterilized and then
crushed so that the Rhizobium colonies therein can be
accessed. Cells from the crushed nodules are plated onto
suitable growth media, i.e. nutrient agar medium, and are
cultivated in the laboratory. These strains can then
proceed through the strain identification process.
Identification of bacterial strains is an evolving
technology at present. At present, most strains of
bacteria are identified and classified based on the
results of a series of tests. The tests include
examinations of morphology, bacterial serology,
quantitative antibiotic sensitivity of the various
strains, phage sensitivity of the strains, and the protein
pattern created by a strain by one or two dimensional
polyacrylamide gel electrophoresis. Any or all of these
techniques may be used together to identify the strains.
The most advantageous technique used by the applicants
here, and considered the best combination of ease and
effectiveness, is one-dimensional gel electrophoresis.
Perferably, large numbers of nodules are collected and
large numbers of Rhizobium cultures are obtained from the
particular locale for the crop in question. For example,
it is appropriate to obtain cultures for hundreds, if not
thousands, of Rhizobium strains from a given geographic
area, and a given crop such as soybean. These thousands
of strains are then analyzed by gel electrophoresis. The
electrophoresis gel patterns are then analyzed. Those
patterns which predominate in the growing areas are
defined, for purposes of this procedure, to characterized
the strains which are naturally predominant. In other
words, the large numbers of gel plates are analyzed and
placed into groups reflecting the groupings of the gel
1335~34
--8--
patterns. The numbers of cultures falling in each group
are then compared to determine which of the groupings, or
strains, is most represented and therefore predominates in
the particular region under consideration. Those isolates
which are found to have identical gel patterns are defined
to be the same strain. It will be discovered that
different frequently isolated strains will be found to be
differentially competitive in different growing regions,
and that certain strains will seem to predominate in any
one localized growing region.
Once the most widely isolated strain in a given
growing region is determined, that strain is considered
the naturally predominant and competitive strain for
purposes of this procedure. In order to fully verify its
competitiveness, and to ensure that the strain has not
previously mutated while in culture, it is then
appropriate to conduct a field test of the strain to
ensure that it nodulates properly and that its
competitive is replicatable in the actual field
environment. Also appropriate is a test for nitrogen
fixing activity. This step can be performed in green
house or phytotron on individual plantlets in pots or
trays. The plants are inoculated with the putative
predominant competitive strain and then are assayed for
acetylene reduction. This not only verifies that the
nitrogen fixing activity of the strain occurs in
conjunction with the selected crop plant, but serves as a
base line for later efforts to select for increased
nitrogen fixing activity.
The confirmation test for competitiveness is a field
test for nodulation activity. This is done by planting
fields of the putative naturally predominant competitive
strain. Controls should include both uninoculated seeds
and plants inoculated with commercial, but presumably
less-competitive, strains. In order to be deemed a
successful naturally predominant competitive strain, the
9 1335434
selected strain should be recoverable from nodules in
inoculated plants at a rate significantly higher than
recoverable from plants which were either not inoculated
or inoculated with non-competitive strains and also at a
rate significantly higher than the commercial strain can
be recovered from nodules..
Strains which can be re-isolated at high frequency
from nodules of legume plants inoculated with the strain
of interest are determined for purposes of experiment to
be naturally predominant and competitive under field
conditions. Thus at this stage of the procedure strains
have been isolated and identified which are indigenous,
but which can also withstand laboratory isolation and be
reintroduced while retaining their competitiveness. In
addition, since by this step we have verified that the
nodulation of the plants is increased by inoculation of
this strain, it is also demonstrated that increased
nodulation can be obtained through the use of this strain,
even without enhanced nitrogen fixing activity.
The next step is to proceed to mutagenize cultures of
the naturally dominant strains. Cultures of the strains
are exposed to a mutagenic agent, such as radiation or a
chemical agent. It is preferred within the present
experiment that the mutagen used is the chemical mutagen
nitrosoguanidine. The bacteria exposed to the mutagen are
transferred and allowed to grow out several times in a
liquid medium. The mutagenized cultures are then spread
on agar plates to obtain individual colonies. Once
individual colonies are established, the colonies are
again transferred to a liquid medium and allowed to grow
out. Samples of each of the grown out colonies can then
be inoculated into a serum vial containing a seedling of
the legume plant to which the Rhizobium is associated.
The seedling is isolated from the environment, such as by
covering with a sterile transparent container, and then is
grown under conditions fostering the growth of the legume
1335434
--10--
plant. After a period of time, an assay for nitrogen
fixing activity, again accomplished by testing for
acetylene reduction activity of the seedlings by gas
chromatography or other similar process is performed. By
comparing the comparative nitrogen fixing activity of the
mutated bacteria with the base line for the naturally
predominant strain, mutagenized progeny which have
enhanced nitrogen fixing activity can be isolated. The
strains having the most promising nitrogen fixing activity
10 can then be returned to culture and grown out to provide
sufficient quantities for additional field testing.
Once high nitrogen fixing candidate mutant strains
have been identified, those strains must then be analyzed
again to ensure that the natural competitiveness has not
15 been lost through the mutation process. Accordingly, all
the candidate strains must again be grown out and must be
individually tested for competitiveness in actual field
tri~ls. Concurrently, nitrogen fixing trials can be
replicated in greenhouse or phytotron to ensure repeatable
20 and reliable enhanced nitrogen fixation capability.
Competitiveness can only be truly assessed in field trials
because of the state of the art of the technology of
replicating competitive field conditions in the greenhouse
or phytotron. Thus the strains are then grown out and
inoculated into the appropriate legume plants in the crop
growing areas. Then the populations of the nodules in the
plants can be examined midseason to determine which
strains of bacteria are populating those nodules. For
those strains which remain competitive and achieve
30 enhanced nitrogen fixing activity, those strains should be
present in the nodules in numbers quantitatively increased
over non-competitive strains. In addition, fields
containing the mutant strains having high nitrogen
fixation and competitive advantages should give enhanced
yield over those fields having no inoculant or other
commercial, non-competitive strain, inoculants available.
1335434
Detailed Description of Example
It was decided to generate novel strains of Rhizobium
japonicum bateria for particular uses as a soybean
inoculant in two major soybean-producing areas of the
United States. As a first step in this process, an effort
was undertaken to identify the natually predominant
strains of R. ~aponicum in the selected states of Iowa and
Louisania. Field teams visted soybean fields in each
state in which no commercial inoculant was applied to the
fields during planting. Nodules were recovered from
soybean plants in the fields. The nodules were surface
sterilized and crushed. Rhizo~ium cell~ isolated from the
nodules were then plated on to a nutrient agar medium, in
which they were cultured and returned to the laboratory.
In the laboratory, cultures of the strains were grown
up and then analyzed using one-dimensional gel
polyacrylamide gel eletrophoresis. Protein gel patterns
were created for each of the Rhizobium isolates obtained
from each state. The gel patterns from the lsolates were
then comparatively analyzed to determine which patterns
were predominant in each of the two designated states.
That anaIysis was able to identify certain gel patterns
which predominated from the samples taken. The gel
patterns were grouped and assigned tenative strain
numbers, nu~bers assigned to the groups of gel patterns to
help group the patterns into related strains.
From the Iowa samples, the strain designated IA2838
was identified, along with one other Iowa strain, as the
most predominant. From the Louisiana samples, a strain
designated LA1304 (ATCC accession no. 53537~ was determined,
again along with one other strain, to be the strain best
represented among the gel patterns. For purposes of these
procedures, both the strains were determined to be
naturally predominant strains.
In order to verify the infective activity of these
-12- 1335 434
Rhizobium strains, a phytotron test of infectivity of each
of these Rhizobium strains was conducted. Bacteria from
each strain was inoculated onto soybean seeds grown up
individually in the phytotron. Acetylene reduction assays
were taken on the young soybean plants to indicate
relative nitrogen fixing activity of the strain when
inoculated into the soybeans. Both of these strains were
found to be infective in soybean and to form nodules in
the roots of soybean plants, and to exhibit reasonable,
though not enhanced, acetylene reduction activity
indicating nitrogen fixation.
To verify that these strains were indeed predominant
and competitive in the locales from which they were
isolated, a field test was conducted using the naturally
predominant strains in actual field conditions. The
strains were used as an inoculant during planting under
normal local soybean growing practices in soybean fields
in both Iowa and Louisiana. To investigate the
differential competitiveness on the Iowa and Louisiana
strains, naturally competitive strains from both locales
were inoculated into fields in both areas. In the field
trials in each state, there were double controls, one
control being plants which were not inoculated
intentionally with any inoculant, and the second control
being planted with a commercially available inoculant from
Nitragin Co. In the Iowa site, the Iowa naturally
predominant strains were identified in 37% of the total
Rhizobial population recovered from nodules of
uninoculated plants. This indicates a population of this
naturally occurring strain sufficient to cause nodulation
in plants even without intentional inoculation. Where the
commercial inoculant was used, bacteria corresponding to
the experimental Iowa naturally predominant strain were
found in 30% of the total Rhizobial population in nodules
recovered. For the plants in which the experimental
inoculant was utilized, 67~ of the Rhizobial population in
1335434
-13-
nodules were found to be the Iowa strains of naturally
predominant bacteria. By contrast, only 5% of the
Rhizobial population in nodules were found to be bacteria
corresponding to the Louisiana naturally predominant
strains, which were also included in the experimental
inoculant. Similar results were obtain in the Louisiana
site where the uninoculated plants had nodules w~ich were
occupied at 33% by the Louisiana naturally predominant
strains, the Nitragin Co. inoculated plants yielded the
10 naturally predominant strains in 13% of the Rhizobial
population in their nodules, and the experimentally
inoculated plants had the naturally predominant strains in
63~ of the Rhizobial population in their nodules. The
experimental inoculant again included populations of both
naturally predominant strains. At the Iowa site, only
about 5% of the Rhizobial population in nodules contained
bacteria of the naturally predominant Louisiana strain,
- with a similar result being reached for the naturally
predominant Iowa strains at the Louisiana site. Thus it
was determined that the naturally predominant strains
effectively nodulated plants in field conditions and were
very competitive with existing indigenous Rhizobium and
with other inoculants to compete for nodulation sites in
soybean roots. In addition, it was also made clear by
this test that for each of the two geographic locales
selected, the naturally predominant strains from those
locales were superior in each locale to the naturally
predominant strain from the other.
Once the competitiveness and nodulation activity of
the naturally predominant strains was verified, the
strains were then mutagenized and selected for high
nitrogen fixation activity. Shown at Figure 1 is a flow
chart of the process used to perform this step. The
chemical mutagen used was nitrosoguanidine. The bacteria
were exposed to the mutagen and then transferred and
allowed to grow in a liquid medium several times. The
_ 1335431
-14-
mutagenized colonies were then spread on agar plates to
obtain individual colonies which were again transferred to
a liquid medium to grow out. Bacteria from the grown out
colonies were placed on soy~ean seedlings grown out from
surfaced sterilized soybean seed grown in a moist sterile
petri plate for three days. The soybean seedling and the
inoculant was placed in a serum vial containing sterile
vermiculite and nitrogen free nutrient solution. The
soybean seedling was covered with a sterile plastic bag
10 and grown in a lighted phytotron for 18 days, after which
the acetylene reduction activity was determined by gas
chromatography. Mutaqenized colonies which showed the
best nitrogen fixing activity (acetylene reduction
activity) were re-inoculated into additional plants and
15 retested to verify that the activity was replicatable.
Shown in Figures 2 and 3 are charts illustrating the
enhancement of nitrogen fixation activity obtained from
the mutagenized strains. Illustrated in Figure 2
demonstrates the relative acetylene reduction activity of
strain IA2838 (ATCC accession no. 53538) compared to
Iowa strain K567 (ATCC accession no. 53535), which is a
mutagenized version of IA2838. The units are arbitrary.
Shown in Figure 3 is a similar chart showing the enhanced
nitrogen fixation activity of strain S258 (ATCC accession
no. 53536) which is a high-nitrogen fixing mutant of
naturally competitive strain Louisiana LA1304.
The mutant strains S2~8 and K567 were then used as
inoculants in actual field tests to attempt to verl~y
competitiveness and increased nitrogen fixation activity
by measuring yield. Each strain was tested against its
30 wild type ancestor. Strain S258 inoculated using a peat
formulation exhibited a 9.9% increase in yield over its
wild type ancestor used similarly as an inoculant in
Louisiana on soybean of variety Centennial. On a similar
test utilizing a peat formulation grown in Illinois on
35 Williams 82 soybeans, no difference in yield was obtained
between the mutant strain and the wild type. Strain K567.
1335~34
-15-
inoculated using a peat formulation, as compared to strain
IA2838, also inoculated using a peat formulation, on
fields of Corsoy*79 and Williams~82 soybeans in fields in
Ohio and Wisconsin exhibited an average yield increase of
13.4%. Field trials of both of these strains were
conducted using uninoculated fields and also against using
an existing commercial inoculant (Nitragin), and both
mutated strains showed statistically significant yield
enhancement over both these controls also.
It is also possible to further enhance the nodulation
competitiveness of these competitive mutant strains. If
the speed with which the Rhizobium begin the nodulation
process can be increased, the first nodulating bacteria
will have a competitive advantage, because they already
occupy the nodules, over other strains. It is already
known that soybean root extract can be used to enhance the
nodulation activity of Rhizobium japonicum strains. This
phenomenon also is effective with the novel strains
produced by the present process.
To verify that effect, soybean seeds were germinated
in soil contained in clear plastic containers. The seeds
were sprouted and, after sprouting, a mark was made on the
container indicating the position of the root tip. The
soil was then inoculated with a viable culture of
Rhizobium japonicum strain K567. The plants were allowed
to grow out and then the nodules in the roots of the
plants were scored, both for number and for location
relative to the root tip marXer. Control plants were
inoculated with untreated K567 bacteria while experimental
plants were inoculated with bacteria pre-treated with a
nutrient broth in which soybean seedlings had been
previously grown.
The results are displayed in Fig. 4 and Table 1
below. Fig. ~ indicates that there were elevated numbers
of nodules overall in the experimental plants and that the
nodules were generally higher on the plant roots,
indicating faster nodule formation. Table 1 verifies this
*Trade Mark
1335434
-16-
result. Therefore, this pre-treatment phenomenon is
effective even for the novel competitive strains created
by this process.
Table 1
Control Pre-Treated
Average distribution of -0.02cm -0.49cm
the Uppermost nodule from
the root tip marker (rtm)
(minus indicates above the
r.t.m.)
Percent of nodulation below 82.4% 84.5%
the r.t.m.
Percent of nodulation on 32.0% 4.9%
lat-eral roots
15 Nodules per plat on primary 1.9 8.3
root
Nodules per plant on lateral 2.7 1.5
root
Thus this example demonstrates that novel competitive
and high nitrogen fixing strains of Rhizobium bacteria can
be created using the process of the present invention.
This approach has two advantages over approaches
previously known in the technology. This approach manages
to select competitive Rhizobium bacteria without having to
attempt to mimic competitive any field conditions in order
to select for strains that would be competitive in the
field. Previous inoculant strains have not been properly
competitive, and the state of the art in mimicing field
conditions to accurately select for competitiveness is not
very sophisticated. In addition, this approach does not
require that the experimenter identify, quantify, or even
study which characteristics of the Rhizobium itself are
necessary or advantageous in making the strain competitive
in the field (ie. able to become a major occupant in
nodules of soybeans or other legumes grown in field crop
conditions). This process takes advantage of the natural
133543~
-17- _
adaptation of Rhizobium activity occurring in agricultural
fields in any event.
