Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
O 94/25568 216 0'~ 8 0 pCT/CA94100243
1 "COLD-TOLERANT STRAINS OF RHIZOBIUM SPECIES'
2 FIELD OF THE INVENTION
3 This invention relates to the identification, selection and use of
novel cold-tolerant strains of Rhizobium species useful for improving
nodulation,
nitrogen fixation and biomass development in legume forage crops at cold soil
5 temperatures.
7 BACKGROUND OF THE INVENTION
s Biological nitrogen fixation is the consequence of an unique,
9 complex symbiosis between Rhizobium bacteria and legume host plants (peas,
to soybeans, alfalfa, sweet clover). The first stage in this process is the
penetration
11 and infection of the host root hairs by rhizobial bacteria, followed by
initiation and
12 development of root nodules. Nodules are specialized plant structures that
13 contain transformed Rhizobium cells, i.e., bacteroids, which are capable of
fixing
14 nitrogen. This stage involves very precise, specific recognition of the
appropriate
symbionts, followed by physical penetration of host root hairs by the
rhizobial
16 bacteria. The rhizobial bacteria then form an infection thread which moves
into
the host root cortex after which, the rhizobial bacteria undergo rapid
18 multiplication. Subsequently, the rhizobial bacteria become pleomorphic,
their
i9 nuclear material degenerates and the resulting bacteroids develop the
enzyme
2 o complexes, particularly nitrogenase, required for nitrogen fixation (Paul,
E.A. and
21 Clark, F.E. (1989) Soil Microbiology and Biochemistry. Academic Press Inc.
pp.
22 273). The environmental, nutritional and physiological conditions required
for
23 rhizobial cell growth and the successful establishment of efficient
nitrogen-fixing
24 symbioses are known (Trinick, M.J. (1982) In W.J. Broughton (Ed.) Nitrogen
2 5 Fixation Vol. 2. Clarendon Press. pp. 76-146).
2 5 The amounts of nitrogen fixed by rhizobia:legume symbioses are
2~ significant and in agricultural situations, can be used to supplement or
replace
2 s nitrogen fertilizer applications (a typical rate of nitrogen fixation by
nodulated
29 alfalfa is up to 250 kg/hectare/year; (Atlas, R.M. and Bartha, R. (1981)
Microbial
3 o Ecology. Addison-Wesly Pub. Co. pp. 560). Consequently, legume crops have
31 become an integral component of most field crop rotations used in
agriculture.
2160~8U
WO 94/25568 PCTlCA94100243
1 In addition to its role in improving crop production, the nitrogen fixed by
legume
2 crops is important as a source of protein in livestock feed. Essentially all
nitrogen
3 fixed by rhizobia:legume associations is in the protein form. Proper
inoculation
with highly effective strains of Rhizobium meliloti results in increased
nitrogen
(i.e., protein) yields of alfalfa forage (Rice, W.A. and Olsen, P.E. (1983)
Can. J.
6 Soil Sci. 63: 541-545). Commercial inoculant formulations are commonly used
when planting legume crops to ensure that sufficient rhizobia are present to
s establish effective nitrogen-fixing systems. Various types of commercial
9 rhizobium inoculant carriers, formulations and preparations are known
including
liquids, clay- or peat-based powders and granules (Thompson, J.A. (1980) In
11 Methods for Evaluating Biological Nitrogen Fixation. F.J. Bergerson (Ed.)
John
12 Wiley and Sons Ltd. pp. 489-533). The carriers may or may not be sterilized
13 prior to inoculation with rhizobial cells.
14 Although Rhizobium bacteria are widely distributed in soils, only
25% of naturally occurring rhizobial strains are considered to be desirable
for
1s incorporation into commercial inoculant formulations (Metcalfe, D.S. and
Elkins,
1~ D.M. (1980) Crop Production: Principles and Practises, 4th Ed. MacMillan
Pub.
18 Co. Inc. pp. 774). Selection of rhizobial strains for production of
commercial
19 inoculants is typically based on: (a) the ability to nodulate the host
legume, (b)
2 o nitrogen fixation performance, (c) adaptability to inoculant production
processes,
21 and (d) efficacy and performance under field conditions (Thompson, 1980).
The
22 most common basis for selection of strains for inoculant development has
been
23 to isolate strains from the root regions or nodules of host plants or from
soils in
2 4 which the host plants were previously grown.
2 5 Although the agronomic and economic benefits resulting from
2 6 establishment of rhizobia:legume symbioses are well known, commercial
2 ~ rhizobial inoculants are not consistent in their efficacy and performance,
and
28 nodulation failures after use of commercial inoculants are common (Miller,
R.H.
2 9 and May, S. (1991 ) In The Rhizosphere and Plant Growth. D.L. Keister and
P.
3 o B. Cregan (Eds.) Kluwer Academic Publishers. pp. 123-134). The primary
31 reasons for inoculant failure are the inability of inoculant strains to out-
compete
32 indigenous rhizobial bacteria for root-infection sites (Amarger, N. (1984)
In
2
__. O 94/25568 '~ ~ ~ PCT/CA94100243
1 Current Perspectives in Microbial Ecology. M.J. Klug and C.A. Reddy (Eds.)
Am.
2 Soc. Microbial. Pub. pp. 300-305.), and/or the inability of the introduced
rhizobial
3 cells to survive in adverse environmental conditions after planting of
inoculated
seed (Lie, T.A. (1974) In The Biology of Nitrogen Fixation. A. Quispel (ed.)
North-Holland Pub. Co. pp. 555-582). Consequently, considerable attention has
6 been focused on improving the modulation and nitrogen fixation performance
of
commercial rhizobial inoculants by: (a) development of novel inoculant carrier
8 substrates and formulations (see for example U.S. Patents 3,616,236;
4,755,468;
9 4,849,005; and 4,875,921 ), (b) selection of superior-performing naturally
occurring rhizobial strains originally isolated from the host crop
(Bourdeleau, L.M.
11 and Antoun, H. (1977) Can. J. Plant Sci. 57: 1071-1075; Bourdeleau et al.,
12 (1977) Can. J. Plant Sci. 433-439; and Rice, W.A. (1982) Can. J. Plant Sci.
62:
s3 941-948), and (c) using genetic engineering technology to improve the
14 modulation or nitrogen-fixation performance of known rhizobial strains
(e.g., U.S.
Patents 4,711,656; 4,713,330; and 5,023,180).
16 The optimum temperature for modulation and nitrogen-fixation by
1~, most rhizobial bacteria ranges between 25 - 30°C (Trinick, 1982).
It is known
18 that decreasing temperatures will significantly reduce and eliminate the
19 modulation ability of rhizobial bacteria that are commonly used for
inoculation of
2 o crop legumes (Gibson, A.H. (1971 ) Plant Soil (Special Vol.): 139-152;
Roughley,
21 R. J. and Dart, P.J. (1970) Plant Soil 32: 518-520). Low temperatures also
22 greatly impair the nitrogen-fixing ability of modulated legumes (Layzell et
al.,
23 (1984) Can. J. Bot. 62: 965-971; Waughman, G.J. (1977) J. Exp. Bot. 28: 949-
2 4 960).
2 5 Although 35°C is the optimum temperature for peak modulation and
2 5 nitrogen fixation by alfalfa inoculated with Rhizobium meliloti (Trinick,
1982), this
2 ~ symbiosis operates effectively and efficiently in a temperature range
between 20-
2 8 25°C (Heichel, G.H. and Vance, C.P. (1983) In Nitrogen Fixation.
Vol. 3.
2 9 Legumes. W.J. Broughton (Ed.) Clarendon Press. pp. 99-143). However, at
3 0 15°C, alfalfa nitrogenase activity is less than 60% of peak
activity, and at 10°C,
31 nitrogenase activity is further reduced to less than 40% of peak activity
(Cralle,
32 H.T. and Heichel, G.H. (1982) Crop Sci. 22: 300-304). Numbers of nodules
and
3
0
WO 94/25568 PCT/CA94/00243
1 weights of nodules formed on alfalfa roots are also greatly reduced at low
2 temperatures (Rice, W.A. and Olsen, P.E. (1988) Biol. Fertil. Soils 6: 137-
140).
3 It is known that some rhizobial species are able to survive and
function in cold temperatures. For example, Prevost et al. (Prevost et al.,
(1987)
Plant Soil 98: 313-324), isolated forty-eight rhizobial strains from three non-
6 agricultural legume species indigenous to the Canadian high arctic. However,
although these strains could be grown in liquid culture at 5°C, they
grew poorly
at 30°C. None of the arctic rhizobia were able to nodulate legume
forage crop
9 plants. Laboratory research performed by Ek-Jander and Fahraeus (Ek-Jander,
1 o J. and Fahraeus G. (1971 ) Plant Soil (Special Vol.): 129-137)
demonstrated that,
11 although strains of Rhizobium trifolii isolated from the sub-arctic were
able to
12 form nodules on clover at 10°C, the process was relatively
inefficient when
13 compared to the nodulation rate at 20°C.
14 Furthermore, the assessment of the field efficacy and performance
of specific rhizobial strains used to inoculate legumes has been difficult
because
16 of the lack of reliable methods to precisely identify and quantify
individual
1~ rhizobial strains. Polyclonal antibody technology has been used to develop
1 s microscopic procedures for observation of rhizobial species on roots and
in soils
19 (Reyes, V.G. and Schmidt, E.L. (1981 ) Plant Soil 61: 71-80.), as well as
other
2 o procedures for detection of rhizobial cells (Pugashetti, B.P. and Wagner,
G.H.
21 (1979) Plant Soil 53: 463-475; Olsen et al., (1983) Can. J. Microbiol. 29:
225-
22 230.). Other approaches have used antibiotic resistance and genetic marking
23 (Stacey, G. and Brill, W.J. (1982) In Phytopathogenic Prokaryotes. Vol. 1.
M.S.
24 Mount and G.H. Lacey (Eds.) Academic Press Inc. pp. 225-247.).
2 5 SUMMARY OF THE INVENTION
2 5 The inventors discovered, surprisingly, that strains of cold-tolerant
2~ Rhizobium species can be detected and screened which can nodulate legumes
2 8 effectively at both cold and moderate soil temperatures. Thus, one aspect
of the
29 invention provides a method for screening a culture collection of one or
more test
3 o strains of Rhizobium species to detect and select cold-tolerant strains
for legume
4
O 94/25568 PCT/CA94100243
1 crop groups, comprising:
2 (a) individually culturing the test Rhizobium species strains
at a
3 temperature of less than about 10C;
4 (b) selecting those Rhizobium strains from step (a) which exhibit
growth;
6 (c) inoculating test legume seeds of the legume crop group with
the
selected strains from step (b);
s (d) germinating the inoculated seeds and growing the test legume
9 plants under controlled conditions where the temperature is
1 o maintained at between about 9 to 15C for a period of time
11 sufficient for nodules to be established on the legume plants;
12 (e) selecting those Rhizobium strains from step (d) for which
the test
i3 legume plants exhibit modulation;
14 (f) inoculating test legume seedlings of the legume crop group
with
the selected strains from step (e) and inoculating control legume
16 seedlings with one or more temperate strains of Rhizobium
1 ~ species;
1 s (g) growing the test and control legume plants under controlled
19 conditions in which the roots are maintained at a temperature
of
less than about 10C while the shoot temperature is maintained
at
21 greater than about 15C, for a time sufficient for the control
plants
22 to establish effective nodules; and
2 3 (h) selecting, as cold-tolerant Rhizobium strains, those Rhizobium
2 4 strains from step (g) for which the test legume plants exhibit
2 5 improved modulation over that exhibited by the control legume
2 6 plants.
2 ~ In specific embodiments of the invention the control legume
plants
2 8 are inoculated with temperate Rhizobium meliloti strains selected
from a group
2 9 consisting of 102F34, BALSAC, URB-165 and NRG-185, and the test
and control
3 o seeds and seedlings are alfalfa or sweet clover.
31 The screening method preferably includes the further steps of
3 2 inoculating second test legume seeds with the selected strains
and inoculating
5
0
WO 94125568 PCTICA94100243
1 second control legume seeds with temperate strains of Rhizobium species. The
2 further steps are as follows:
3 (i) inoculating second test legume seeds of the legume crop group
with the selected strains from step (h) and inoculating second
control legume seeds with one or more temperate strains of
6 Rhizobium species;
(j) growing legume field crops from the inoculated test and control
legume seeds; and
9 (k) confirming as cold-tolerant strains of Rhizobium species, those
1 o strains from step (j) for which the test field crop exhibits improved
11 nodulation or plant biomass development over that obtained for the
12 control field crop.
13 The screening method provides novel biologically pure cultures of
14 cold-tolerant strains of Rhizobium. This invention also extends to a
biologically
z5 pure culture of a cold- tolerant strain of Rhizobium meliloti, NRG-34 (ATCC
#
16 55340).
z ~ Further broad aspects of this invention include a method of
18 promoting growth of legume crop groups by inoculating legume seeds of the
19 legume crop group with an agriculturally effective amount of the novel cold-
2 o tolerant strains of Rhizobium species, and novel agricultural inoculant
21 compositions containing the novel strains in admixture with an inoculant
carrier
2 2 medium.
2 3 This invention further provides a monoclonal antibody whenever
24 produced using, as immunogens, the novel cold-tolerant strains of Rhizobium
2 5 meliloti. This invention also provides a method for detecting or
quantifying cold-
2 5 tolerant Rhizobium meliloti strains in test samples of culture media,
nodule
2 ~ tissue, inoculant compositions and soil, comprising assaying the test
sample with
2 8 these monoclonal antibodies.
2 9 In specific embodiments monoclonal antibody MAb-7 is produced
3 o using Rhizobium meliloti strain NRG-34 (ATCC No. 55340) as the immunogen
3 z and NRG-F12P92F as the hybridoma cell line. This invention also provides a
32 method for detecting or quantifying cold-tolerant Rhizobium meliloti strain
NRG-
6
D 94/25568 PCTlCA94100243
1 34 (ATCC No. 55340) in test samples of culture media, nodule tissue,
inoculant
2 compositions and soil, comprising assaying the test sample with MAb-7. In a
3 particular embodiment assays for the cold-tolerant strains of Rhizobium
meliloti
selected by the method described in this invention or NRG-34 (ATCC No. 55340)
are carried out by an ELISA assay or an immunoblot assay.
6 While this invention is demonstrated by work conducted with test
strains of Rhizobium meliloti using alfalfa as the test and control legume
crop
s group, the invention is not so limited. Other legume crop groups may be used
to detect and select cold tolerant strains of R. meliloti, for instance sweet
clover.
1 o Cold tolerant strains of Rhizobium trifolii may be detected by the
screening
11 process, in which case the test and control legume crop group is preferably
red
i2 clover. Similarly, cold tolerant strains of Rhizobium leguminosarum may be
13 detected and selected, preferably using peas or lentils as the test and
control
14 legume crop group. Similarly, cold tolerant strains of Rhizobium phaesolii
may
be selected and detected, preferably using beans as the test and control
legume
16 crop group. Similarly, cold tolerant strains of Bradyrhizobium japonicum
may be
1~ detected and selected using soybeans as the test and control crop group.
.18 BRIEF DESCRIPTION OF THE DRAWINGS
19 Figure 1 is a plot of doubling times of Rhizobium meiiloti strains
2 o cultured at 10°C and the numbers of nodules formed on alfalfa
seedlings during
21 2 weeks growth at 10°C; and
22 Figure 2 is a bar graph illustrating the reactivity of MAb-57 with
2 3 Rhizobium melilofi and Rhizobium leguminosarum bv. trifolii strains.
24 DESCRIPTION OF THE PREFERRED EMBODIMENT
2 5 The present invention relates to methods of screening a culture
2 6 collection of strains of Rhizobium species, selecting as cold-tolerant
strains those
2 ~ strains of Rhizobium which are capable of growth and modulation at low
2 8 temperatures, and using the cold-tolerant Rhizobium strains to inoculate
legume
2 9 crops to increase modulation, nitrogen fixation and biomass development.
While
3 o the screening process is demonstrated herein by detecting and selecting
cold
216y ?8Q
WO 94/25568 PCT/CA94/00243
1 tolerant strains of Rhizobium meliloti, using alfalfa as the test and
control legume
2 crop group, the screening process has applicability in detecting and
selecting
3 cold tolerant strains of other Rhizobium species including R. leguminosarum,
R.
phaesolii, R. trifolii, and Bradyrhizobium japonicum. The preferred matching
of
Rhizobium species with legume crop groups is as follows:
5 Rhizobium species screened Legume Crop Group
R. meliloti alfalfa, sweet clover
s R. leguminosarum peas, lentils
R. phaesolii beans
1 o Bradyrhizobium japonicum soybeans
11 R. trifolii red clover
12 Hereinafter, the invention is illustrated with reference to R. melilotiand
alfalfa, but
13 is demonstrative of utility of the invention with other Rhizobium species
and other
14 legume crop groups.
The present invention also relates to methods for production of
15 monoclonal antibodies which react specifically with selected cold-tolerant
1~ Rhizobium meliloti immunogen strains, and the use of said monoclonal
18 antibodies to detect and quantify the selected cold-tolerant Rhizobium
meliloti
s9 immunogen strains in plant samples, soil samples, inoculant compositions,
and
laboratory cultures.
21 As used herein, the term "cold-tolerant" in the context of the
22 Rhizobium strains refers to the ability of these microorganisms to grow in
culture
23 at temperatures less that about 10°C, to nodulate legume root tissue
at
24 temperatures less than about 10°C, for example at about 5-
9°C, and to improve
2 5 plant growth.
2 6 As used herein, the term "temperate" in the context of the
2 ~ Rhizobium strains refers to those microorganisms which show an optimal
ability
2 8 to nodulate legume root tissue at temperatures in the range of about 20 -
35°C
2 9 and to improve plant growth, but which show limited ability to nodulate at
3 o temperatures less than 10°C.
8
.-O 94125568 PCT/CA94/00243
1 As used herein, the term °biologically pure" refers to a degree of
2 chemical purity wherein impurities that substantially effect the functional
3 properties of the purified component have been substantially removed.
As used herein, the term "agriculturally effective amount" refers to
an amount of an active agent that is sufficient to produce the desired
agricultural
5 effect. For example, in the case of Rhizobium melilofi, an "agriculturally
effective
amount" of Rhizobium meiiloti would be the amount necessary to provide
s nodulation and improve plant growth.
g As used herein, the term "improved nodulation" refers to an
1 o increase in the number of effective nodules and/or an increase in the
weight of
11 effective nodules on test plants as compared to control plants.
12 As used herein, the term "improved nitrogen fixation" means an
13 increase in nitrogen content, as expressed as % nitrogen, of plant tissues
from
14 test plants as compared to control plants, and/or an increase in the
protien yield,
as expressed as kg of protein per hectacre, of test plants as compared to
control
16 plants.
As used herein, the term "improved plant biomass development"
18 means an increase in the dry weight of root biomass and/or shoot biomass of
19 test plants as compared to control plants.
2 o The present invention discloses a method for the identification,
21 selection and use of cold-tolerant strains of Rhizobium species that are
capable
22 of efficient metabolism, nodulation and nitrogen fixation at low
temperatures. This
2 3 method preferably includes five stages which are characterized
hereinbelow, with
24 references to R. meliioti and alfalfa.
2 5 Stage 1 - Screening for Growth
2 6 A special semi-automated miniaturized microtiter plate technique
2 ~ is used to screen large numbers of pure cultures from a collection of
Rhizobium
2 s meliloti strains. The individual strains are preferentially incubated in 4-
ml screw-
2 9 cap vials containing 2 ml of sterile yeast extract mannitol broth (YEMB)
at 30°C
3 o for 3 days. The individual cultures can also be cultured in larger volumes
of liquid
31 culture, or alternatively, grown on agar. Then, 10 p.l of an individual
culture are
9
16~~ ~~p
WO 94/25568 ' PCT/CA94/00243
1 added to 150 p.l of sterile YEMB in a well in 96-well microtiter plates. The
2 microtiter plates are then placed into an incubater at 10°C for 140
hours. The
3 plates are removed at 24-h intervals and the optical density of the broth in
the
individual wells is determined with an ELISA plate reader. Alternatively, the
miniature microtiter plate technique can be modified by using microtiter
plates or
6 tissue culture plates with larger wells. For example, the individual
cultures can
be grown at 25°C for 4 days in erlenmeyer flasks containing 50 ml YEMB.
Then
s 1 ml of the resulting culture is diluted with 4 ml of sterile YEMB, mixed
well and
9 then 50 p.l of the diluted culture added to 1000 ~.I of sterile YEMB in each
well
of a 24-well tissue culture plate. A sterile glass bead is added to each well.
The
11 24-well plates are then placed into a pan covered with a styrofoam lid and
then,
12 submerged into a shaking water bath. The water bath temperature is adjusted
13 so that the temperature inside the wells of the plate is 10°C. The
plates are
14 incubated for 234 h; the plates are removed periodically for determination
of the
i5 optical density of the culture (an indication of growth) in each well. At
the
16 completion of the incubation period, the growth in each individual well is
tested
1~ with an ELISA test following the technique of Olsen et al. (1983) and using
1s polyclonal antibodies prepared for Rhizobium meliloti, to confirm that the
culture
19 is biologically pure and a strain of Rhizobium meliloti. Preparation of the
2 o polyclonal antibodies that specifically react with Rhizobium melilofi
strains are
21 prepared following the technique of Olsen et al. (1983).
22 Stage 2 - Screening for Nodulation
2 3 Cultures that test positive and biologically pure are used to
24 inoculate alfalfa seed which are then placed into growth pouches and then
2 5 incubated in a growth chamber held at a temperature between about 9 and
15°C,
2 6 preferably constant at 10°C or 15°C with a 16h light and 8h
dark diurnal cycle.
2 ~ Nodules are counted at 2 weeks and 4 weeks, after which the plants are
2 8 removed from the growth pouches.
2160780
.i0 94/25568 PCT/CA94/00243
1 Stage 3 - Comparin4 Test Strains with Control Strains in the Laboratory
2 Rhizobium melilofi strains which demonstrate the ability during the
3 Stage 1 process, to grow in liquid culture at 10°C and are also able
to nodulate
4 alfalfa grown at 10°C or 15°C (Stage 2), are further screened
with the method
outlined below. Alfalfa seeds are germinated in growth pouches (5 seeds/pouch)
6 submerged in a water bath so that the root and ambient temperatures are at
21 °C. After the alfalfa seedlings are established at 21 °C, the
growth pouches are
s transferred to recirculating cold-water baths with temperatures held at 9,
10, 11
or 12°C for 2 hours to allow the root-zone temperature to equilibrate.
Then, the
1 o test alfalfa seedlings are inoculated individually with the test cold-
tolerant strains
11 of Rhizobium meliloti selected with the Stage 1 and 2 methods. The inocula
are
12 1-ml aliquots (1X108 cells/ml) of 4-day-old liquid cultures grown in YEMB
at 15°C.
13 The performance of the test cold-tolerant strains are compared to
uninoculated
14 alfalfa seedlings and alfalfa seedlings inoculated with temperate Rhizobium
meliloti strains used in commercial alfalfa inoculant products, grown under
the
16 same conditions. After growth for 6 weeks, the plants are harvested by
removing
1~ them from the growth pouches, excising, counting and weighing nodules, and
18 then determining the dry weights of the plant tissues. The strains for
which the
i9 test alfalfa plants show improved nodulation over that exhibited by
temperate
2 o Rhizobium meliloti strains used in commercial noculants are selected as
cold-
21 tolerant Rhizobium meliloti strains.
22 Staae 4 - Comparing Test Strains with Control Strains in the Field
2 3 The effects on nodulation, nitrogen fixation and plant productivity,
2 4 of the test cold-tolerant Rhizobium meliloti strains from Stage 3 which
2 5 demonstrated an ability to form effective nitrogen-fixing nodules on
alfalfa grown
2 6 at 9, 10, 11 and 12°C, are preferably compared under field
conditions with
2 ~ temperate Rhizobium meliloti strains used in commercial alfalfa inoculant
2 8 products. The test cold-tolerant strains and the temperate commercial
inoculant
2 9 strains are prepared in the same inoculant carriers and formulations,
e.g., liquid,
3 o powdered peat, granules, and are inoculated onto alfalfa seed using
conventional
31 methods. Uninoculated seeds are used as the controls. The seeds are planted
11
WO 94/25568 PCTICA94100243
1 in field plots which are maintained and sampled for two growing seasons.
2 Typically, the field plots are sampled at: (a) the end of the first growing
season,
3 preferably during the month of September, (b) after the onset of the second
4 growing season, preferably during the month of June, and (c) at the end of
the
second growing season, preferable during the month of September. At each
6 sampling time, a 30-cm row of alfalfa plants is dug from each treatment, the
roots are washed, nodules counted and weighed, arid strain nodule occupancy
s is determined. As well, the alfalfa from each treatment row is cut in the
fall of the
9 planting season, and in the following spring and fall for determination of
hay
1 o production. Strains are confirmed as cold-tolerant if they exhibit
improved
11 nodulation and/or improved plant biomass development over the results from
the
12 commercial inoculant strains.
13 It has been found that cold-tolerant Rhizobium meliloti strains
14 detected and selected with the described process, can be incorporated into
current commercial inoculant carrier compositions, including but not limited
to
15 liquids and peat powders, without impairing their nodulation and nitrogen
fixation
1 ~ processes.
is It was also surprisingly found that a unique cold-tolerant strain of
19 Rhizobium meiiloti, NRG-34 (ATCC No. 55340), in addition to forming nodules
2 o and fixing nitrogen at cold temperatures, performs as well as or
outperforms
21 temperate Rhizobium meliioti strains currently used in commercial alfalfa
22 inoculant products, under temperate crop production conditions by
increasing
23 forage yields (plant biomass) and/or production of higher-protein forage.
24 The cold-tolerant Rhizobium meliloti NGR-34 (ATCC No. 55340),
is deposited with the American Type Culture Collection, Rockville, MD, 20852,
2 5 pursuant to the provisions of the Budapest Treaty on the International
2 ~ Recognition of the Deposit of Microorganisms for the Purposes of Patent
2 8 Procedure, under accession number ATCC-55340.
12
:.O 94/25568 PCTICA94/00243
1 Stage 5 - A Method for Detecting Cold-Tolerant Rhizobium meliloti Strains
2 The present invention also discloses a method for the production
3 of unique monoclonal antibodies (MAb) that are specific to individual
strains of
Rhizobium meliloti and in particular, a monoclonal antibody specific to
Rhizobium
meliloti strain NRG-34 (ATCC No. 55340). Cells of a selected Rhizobium
meliloti
5 strain are killed by steaming, then are emulsified (1:1 ) with Freund's
complete
adjuvant and injected intramuscularly (0.10 ml) into BALB/c mice. After six
8 weeks, a series of three booster antigen injections with killed cells of the
selected
9 Rhizobium meliloti strain emulsified in incomplete Freund's adjuvant, are
1o administered at 10-day intervals. Ten days after the final booster
injection, the
11 spleen cells are harvested from the mice and fused with plasmacytoma cells
(cell
12 line P3-NS1-AG4-1) using polyethylene glycol at pH 8. The fusion products
are
13 then distributed among the wells of 96-well microtiter plates
containing,tissue
14 culture media (pH 7.2) amended with aminopterin. The fusion product plates
are
incubated in a C02 atmosphere for 10 days and then, are weaned from the
15 aminopterin by successive additions of aminopterin-free medium. Viable
1~ hybridomas found in the fusion plate wells are screened for antibody
production
18 and antibody specificity by indirect ELISA testing of the culture media
removed
19 from the hybridoma-bearing culture wells. Hybridomas producing antibodies
with
2 o a high specificity for the immunogenic Rhizobium meliloti strain and no
reactivity
21 with other Rhizobium meliloti strains, are cloned by limit dilution
techniques,
22 increased by culture in larger vessels, and then preserved by freezing in
liquid
2 3 nitrogen. Production of monoclonal antibodies for routine use is done by
24 intraperitoneal injection of actively growing hybridoma cells into BALB/c
mice,
resulting in the production of ascites fluid containing the monoclonal
antibody.
2 5 The monoclonal antibodies are then purified from the ascites fluid by
column-gel
2 ~ chromatography or ammonium sulfate precipitation. Purified monoclonal
2 g antibodies are characterized to class and isotype using commercial
antibody
2 9 typing kits.
3 o The present invention also discloses methods for the use of these
31 monoclonal antibodies for detection and quantification of specific
individual
32 strains of Rhizobium melilofi in alfalfa nodules with a test referred to as
the
13
CA 02160780 1998-09-21
1 "Nodule Occupancy Assay". Alfalfa plants are carefully dug from the soil and
the
2 roots gently washed free of soil. The nodules are then excised with a
scalpel and
3 frozen until analysis. The analysis is initiated by placing frozen nodules
into small
4 test tubes (1 nodule/tube) containing 1.0 ml of phosphate-buffered saline
(PBS)
and then crushing them with a glass rod. One-hundred NI of the crushed nodule
6 suspension is placed into the wells of a microdilution plate, covered and
allowed
'7 to incubate for 12 - 18 h at 4°C. The microdilution plates are then
washed five
8 times in an automated plate-washer using PBS containing 0.05% Tween 20T"~
9 (PBST). An ascites fluid preparation of a selected MAb (e.g., MAb-7) is
diluted
(1:6000) in PBST and mixed well, after which, sheep-anti-mouse Ig biotin
ii conjugate (SAMBi) is added to the diluted Mab suspension at a dilution of
z2 1:8000 and mixed well. The Mab-SAMBi suspension is then added to the
13 washed microdilution plates and incubated for 1.5 h at room temperature.
The
14 plates are then washed 5 times with PBST. Streptavidin-horseradish
peroxidase
conjugate (1:10000 in PBST) is added to the microdilution plates (100 pl/well)
and
16 incubated for 20 min at room temperature after which, the plates are washed
1~ 5 times with PBST. Enzyme substrate (tetramethylbenzidine and hydrogen
18 peroxide) is then added to each well (100 pl/well)and incubated for 30 min.
The
19 enzyme reaction is stopped by the addition of 50 NI/well of sulfuric acid
(2 N).
2 o The plates are then read with an automated ELISA plate reader set at a
21 wavelength of 450 nm. A positive reaction, i.e., binding of a monoclonal
antibody
22 to its antigen, will result in the formation of a bright yellow colour in
the wells
23 containing the antibody-binding strain, whereas wells containing other
strains will
24 remain colourless.
2 5 The present invention also discloses methods for the use of these
2 6 monoclonal antibody preparations for the detection and quantification of
specific
2'7 individual strains of Rhizobium meliloti in inoculant formulations or in
soil
2 8 samples, with a test referred to as the "Immunoblot Assay". Inoculant
29 formulations or soil samples are serially diluted and plated onto
appropriate agar
3 o media in petri plates, then incubated for 48 h at 30°C. Agar plates
containing 30 -
31 300 colonies are selected for testing with the Immunoblot Assay. Circular
32 nitrocellulose membranes are "pre-blocked" by soaking them for 1 h in a PBS
14
21 ~0 780
O 94!25568 PCTICA94/00243
1 ~ solution containing 2% skim milk powder after which, the membranes are
rinsed
2 and washed with PBS. A pre-blocked membrane is then placed over the surface
3 of a petri dish so that the membrane surface is in contact with all of the
microbial
4 colonies that have grown on the agar surface. The membrane is then
immediately removed from the agar plate and washed rigourously so that all
6 visible bacterial residue is removed from the membrane. The membrane is then
dried in a stream of flowing air after which, the membrane is soaked in
acidified
s PBS (pH 2.4) for 20 min to denature any endogenous alkaline phosphatase
9 which may have bound non-specifically to the membrane followed by washing
1 o until the pH is neutral. A selected monoclonal antibody is mixed with
SAMBi
11 conjugate (1:3500 dilution of each component) in PBST containing 1 % fetal
calf
12 sera (PBST-FC) after which, 20 ml of the resulting suspension is
transferred to
13 an empty glass petri dish. The washed membrane is immersed in the
monoclonal
14 antibody-SAMBi-PBST-FC mixture for 1 h at room temperature and then
rewashed in PBST. The membrane is then immersed in streptavidin-alkaline
16 phosphatase conjugate (1:3750 in PBST-FC) for 15 min followed by a thorough
1 ~ wash with PEST and then, immersed for 30 min in a precipitating substrate
for
1 s alkaline phosphatase (i.e., 5-bromo-4-chloro-3-indole phosphate plus
nitroblue
19 tetrazolium). Areas on the membrane that had contacted bacterial colonies
2 o containing the specific antigen for the selected monoclonal antibody turn
dark
21 purple while the rest of the membrane remains uncoloured.
22 Further specific embodiments of this invention are illustrated by the
2 3 following non-limiting examples.
24 EXAMPLE 1
2 5 A culture collection consisting of 226 strains tentatively identified
2 6 as Rhizobium melilofi, was screened to determine the ability of the
strains to
2 ~ grow at cold temperatures. The culture collection was comprised of: (a)
180
2 8 strains isolated from soil and alfalfa root samples collected from the
Peace River
2 9 region of Alberta, Canada between 1970 and 1988, (b) 35 strains isolated
from
3 o soil samples collected between Beaverlodge, Alberta, Canada and Fairbanks,
31 Alaska, USA, in 1988, and (c) 11 temperate Rhizobium meliloti strains used
in
21647 ~0
WO 94/25568 ~ PCT/CA94/00243 _
1 the manufacture of commercial inoculant products. Each strain in this
collection
2 was tested as described hereinabove with the semi-automated miniaturized
3 microtiter plate assay at 10°C for 140 h/assay. At the end of this
assay, an
ELISA test using a non-specific Rhizobium meliloti polyclonal antibody and
following the technique of Olsen et al. (1983) was run on each culture that
grew
6 at 10°C in the microtiter plate assay. The polyclonal antibodies that
specifically
reacted with Rhizobium meiiloti strains were prepared following the technique
of
s Olsen et al. (1983). Any culture that did not test positive in the ELISA
test was
9 either purified by repeated sub-culturing on agar, or was discarded from
further
evaluations.
11 The results of the microtiter plate assay and the polyclonal ELISA
12 test indicated that only 97 strains of the 226 tested, grew in liquid
culture at 10°C
13 and were identified positively as R, meliloti. After 140 h incubation at
10°C, the
14 rate of growth, i.e., doubling time, of these strains was determined based
on the
1 s changes in optical density of the broths in the separate wells of the
microtiter
16 plates. One strain had a doubling time of less than 20h, 2 strains had
doubling
1~ times of between 20 - 40h, 15 strains had doubling times of 40 - 50h, while
the
18 doubling times of the remaining strains were greater than 50h at
10°C (Table 1 ).
19 TABLE 1: Doubling times for Rhizobium meliloti strains
2 o grown in liquid broth at 10°C for 140h.
21
2 2 Doubling time (h) No. of strains
23
2 4 Less than 20 1
2 5 20 - 40 2
2 6 40 - 50 15
2 ~ Greater than 50 79
2 s Total 97
29
16
O 94/25568 PCT/CA94I00243
2~ ss 7ss
1 EXAMPLE 2
2 The relationship between doubling times for 122 Rhizobium meliloti
3 strains grown at 10°C, including 11 temperate Rhizobium meliloti
strains used in
commercial inoculants, and their ability to form nodules at 10°C was
assessed
by determining: (a) strain doubling times with the 24-well microtiter plate
assay,
5 and (b) numbers of nodules formed on alfalfa seedlings grown in growth
pouches
with root zone temperatures maintained at 10°C. Of the 117 strains
tested, 16
8 strains had doubling times less than 24h and also produced more than 2
nodules/plant (Figure 1 ).
EXAMPLE 3
11 The sixteen best-performing test strains from Example 2 were
12 further evaluated for their modulation and nitrogen fixation performance
when
13 inoculated onto alfalfa seedlings grown at root zone temperatures of 9, 10,
11,
14 and 12°C. Four temperate Rhizobium meliloti strains (i.e., 102F34;
BALSAC;
URB-165; NRG-185) used in the manufacture of commercial alfalfa inoculants
15 were also inoculated on to alfalfa seedlings grown at root zone
temperatures of
9, 10, 11, and 12°C for comparisons. Strains 102F34 and BALSAC are used
in
18 NITRAGIN~" formulations manufactured by LiphaTech Inc. (Milwaukee,
19 Wisconsin), strain URB-165 is used in RHIZO-STIK~" and URBANA'~''"
2 o formulations manufactured by Urbana Labs Inc. (St. Joseph, Missouri),
strain
21 NRG-185 is used in N-PROVE" manufactured by Philom Bios Inc. (Saskatoon,
22 Saskatchewan). Pure cultures of the Rhizobium meliloti strains used in
2 3 commercial inoculants were supplied as follows: 102F34 by Dr. S. Smith
24 (LiphaTech Inc.), BALSAC by Dr. L. Bordeleau (Agriculture Canada Research
Station, Ste. Foy, Quebec), URB-165 by Dr. T. Wacek (Urbana Labs Inc.), NRG-
2 6 185 from inventor Dr. Rice's culture collection.
2'7 Alfalfa seedlings were established in growth pouches (5
2 8 seedlings/pouch) held at 21 °C in a growth chamber. Then, the
growth pouches
29 were placed into recirculating water baths held at temperatures set to
maintain
3 0 the root zones at constants of 9°C, 10°C 11 °C, or
12°C. The ambient shoot
31 temperatures were held at 21°C. After a 2-h equilibration period,
the seedlings
m
WO 94/25568 PCT/CA94/00243
1 in each growth pouch were inoculated with 1-ml aliquots (1X108 cells/ml) of
4-
2 day-old Rhizobium meliloti test and temperate strain cultures grown at
15°C.
3 Each temperature treatment consisted of 5 inoculated growth pouches/strain
and
uninoculated growth pouches (controls). The plants were then incubated for 6
5 weeks after which, the following measurements were made: number of
5 nodules/pouch, fresh weight of nodules/pouch, dry weight of roots/pouch, and
dry
weight of shoots/pouch.
8 Nodules were classified as "Effective" or "Ineffective" nodules based
on their physical appearance. "Ineffective" nodules were very small with a
distinct
"white" or a "greenish-white" coloration. "Effective" nodules were
considerably
11 larger than "noneffective nodules, and were pigmented a distinct reddish-
pink
z2 tinge. The pink coloration of the "effective" nodules was due to the
presence of
13 laghaemoglobin, a molecule essential for the nitrogen fixation process.
14 At 9°C cold-tolerant strains NRG-34, NRC-55, NRG-61, NRG-43
and NRG-47 showed improved nodulation over the temperate Rhizobium melfloti
15 strains in terms of the numbers of effective nodules formed on alfalfa
roots. At
1~ 10°C, NRG-34 showed improved nodulation over temperate Rhizobium
meliloti
18 strains in terms of the numbers of effective nodules formed on alfalfa
roots.
19 There were no significant differences in the numbers of effective nodules
formed
2 0 by the strains at 11 °C and 12°C. These results are
presented in Table 2.
18
., O 94/25568 ~ PCTICA94/00243
1 Table 2: Number of effective nodules formed by Rhizobium
2 meliloti strains at cold root temperatures.
3
Root Temperature (°C)
Strain # 9 10 11 12
Uninoculated
control 0 0 0 0
NRG-24 0.3 11.6 ~.7 11.2
N RG-34 2.1 17.8 11.5 11.8
NRG-38 0 1.1 2.6 2.1
NRG-43 3.2 4.8 12.0 9.7
N RG-47 1.8 6.4 13.6 12.6
N RG-55 2.4 6.0 11.9 12.4
NRG-61 3.4 11.6 8.1 8.2
NRG-70 0.4 7.4 13.9 8.9
N RG-79 0.8 10.0 11.2 12.1
NRG-84 0.6 3.5 8.0 1.4
NRG-85 1.1 0.8 4.4 1.5
NRG-133 0 0 5.1 1.2
NRG-233 0.8 7.1 9.5 12.2
NRG-255 0.6 8.0 8.2 9.5
NRG-282 0 5.2 2.6 1.4
N RG-348 0.8 5.2 12.0 11.5
102F34'' 0.8 8.5 18.0 11.4
BALSAC+ 0.4 9.6 11.3 9.8
URB-165+ 0.4 9.4 21.3 12.5
NRG-185+ 1.2 13.2 7.9 15.2
Commercial
strain mean 0.7 10.2 14.6 12.2
Commercial inoculant strain
34 At 9°C, NRG-34, NRG-55, NRG-61 and NRG-43 showed improved
3 5 modulation over temperate Rhizobium meliloti strains in terms of the fresh
weight
36 of efffective nodules formed on alfalfa roots. At 11°C and
12°C, NRG-34 showed
3 ~ improved modulation in terms of the fresh weight of effective nodules over
that
3 s of the temperate Rhizobium melilofi strains. These results are presented
in
3 9 Table 3.
19
WO 94/25568 PCTICA94/00243 _
1 Table 3: Fresh weights (mg/pouch) of effective nodules
2 formed by Rhizobium meliloti strains at cold root
3 temperatures.
4
Root
Temperature
(C)
6 __________________________________
________________~______-
Strain # 9 10 11 12
8
9 Uninoculated
control 0 0 0 0
11 NRG-24 3.4 17.5 23.8 16.0
12 NRG-34 8.7 18.8 30.7 28.2
13 NRG-38 4.9 20.9 28.9 21.5
14 NRG-43 5.3 9.5 22.5 20.7
NRG-47 4.5 15.4 26.4 22.3
16 NRG-55 7.0 16.7 24.2 24.9
17 N RG-61 7.4 20.0 23.9 24.6
18 NRG-70 5.0 19.2 25.7 24.6
19 NRG-79 4.6 16.3 21.4 17.9
2 0 N RG-84 3.5 12.5 18.2 16.6
21 NRG-85 4.7 15.6 27.7 17.1
22 NRG-133 0.4 0.9 15.3 6.3
2 3 N RG-233 3.5 18.1 27.4 26.6
2 4 N RG-255 7.4 22.6 31.2 25.9
2 5 N RG-282 0.1 13.0 8.6 7.8
2 6 N RG-348 4.6 11.6 20.5 14.4
27 102F34+ 3.3 18.4 25.0 17.8
2 8 BALSAC+ 4.9 17.8 29.2 21.3
2 9 U RB-165+ 4.0 22.2 24.9 29.9
30 NRG-185+ 4.6 18.8 25.9 24.7
31 Commercial
32 strain mean 4.2 19.3 26.3 23.4
33
34
3 5 + Commercial inoculant
strain.
3 6 Significant differences in the dry weight of roots in response to
3 ~ inoculation with the different strains, were not observed. However, at
9°C, alfalfa
3 8 plants inoculated with Rhizobium meliloti strains NRG-34, NRG-43, NRG-55,
39 NRG-79 showed improved plant biomass production in terms of plant shoot dry
4 o weight over those plants inoculated with temperate Rhizobium meliloti
strains.
41 At 12°, plants inoculated with NRG-34 showed increased biomass
development
42 in terms of shoot dry weights by 21 % over that of plants inoculated with
~. O 94/25568 ~ PCTlCA94/00243
s temperate Rhizobium meliloti strains. These results are presented in Table
4.
Table 4: Dry weight (mg/pouch) of shoots from alfalfa
inoculated with Rhizobium meliloti strains at
cold root temperatures.
Root Temperature (°C)
Strain # 9 10 11 12
Uninoculated
control 12.9 17.6 16.3 25.0
NRG-24 15.5 41.2* 37.4* 36.0
NRG-34 17.0* 37.5* 31.7* 63.0*
NRG-38 11.8 20.6 20.8 33.0
NRG-43 19.1* 23.4 39.5* 58.9*
NRG-47 17.6 23.7 29.4 46.6*
NRG-55 17.9* 35.0* 30.1 * 50.7*
NRG-61 14.1 26.4 29.6 43.1
NRG-70 14.3 38.6* 42.0* 42.9*
NRG-79 19.2* 36.9* 41.4* 38.7
NRG-84 16.7 20.1 21.2 28.1
NRG-85 16.6 18.9 35.6* 32.7
NRG-133 12.3 18.1 28.4 25.8
NRG-233 15.2 32.4* 39.8* 43.2*
NRG-255 15.3 40.1 * 44.4* 44.8*
NRG-282 15.8 25.6 17.0 28.1
NRG-348 16.1 34.2* 38.2* 42.0*
102F34+ 12.1 38.1 * 40.8* 56.5*
BALSAC+ 16.5 34.2* 49.4* 57.6*
URB-165+ 14.7 38.1 * 42.8* 54.6*
NRG-185+ 16.1 41.6* 49.0* 39.3
Commercial
strain mean 14.9 38.0 45.5 52.0
3 s * Significantly different from the uninoculated control (P<0.05)
3 6 + Commercial inoculant strain
37 EXAMPLE 4
3 s A prototype commercial peat-based inoculant formulation was
3 9 prepared with a cold-tolerant Rhizobium meliloti strain (NRG-34). Dark
sedge
4 o peat was dried to a moisture content of about 12% after which, sufficient
calcium
41 carbonate powder was blended into the peat to adjust the pH to about 6.5.
The
42 peat was then milled to produce a powder which would pass through a 200-
mesh
21
WO 94/25568 PCT/CA94I00243
1 screen. The milled peat was dispensed into plastic bags (80 g/bag) which
were
2 heat-sealed, then sterilized by irradiation. Rhizobium meliloti strain NRG-
34 was
3 grown in a fermenter vessel containing a medium consisting of sucrose (12
g/I),
autolyzed yeast extract (3 g/I), magnesium sulfate (0.2 g/I), sodium chloride
(0.1
g/I), mono-basic potassium phosphate (0.292 g/I), di-basic potassium phosphate
6 (0.193 g/I). The fermentation was performed under controlled temperature,
aeration and pH conditions until the culture reached the late stationary phase
of
8 the growth cycle. The fermenter culture was then diluted to a concentration
of 1
X 109 cells/ml by the addition of fresh sterile fermenter medium and injected
1 o asceptically into each bag containing irradiated peat, in sufficient
volume to bring
11 the final moisture content of the peat formulation to about 40%. The
rhizobial
12 culture was mixed into the peat carrier by kneading each bag several times
after
13 which, the culture was cured for 2 weeks at 25°C. After the 2-week
curing period,
14 the titre of Rhizobium meliloti strain NRG-34 in the peat formulation was 2
X 109
cells/gram.
16 EXAMPLE 5
1~ A prototype commercial liquid-based inoculant formulation was
18 prepared with a cold-tolerant Rhizobium meliloti strain (NRG-34). Rhizobium
19 meliloti strain NRG-34 was grown in a fermenter vessel containing a medium
2 o consisting of sucrose (12 g/I), autolyzed yeast extract (3 g/I), magnesium
sulfate
21 (0.2 g/I), sodium chloride (0.1 g/I), mono-basic potassium phosphate (0.292
g/I),
22 di-basic potassium phosphate (0.193 g/I). The fermentation was performed
under
23 controlled temperature, aeration and pH conditions until the culture
reached the
24 late stationary phase of the growth cycle. The liquid formulation was then
2 5 prepared by first adjusting the optical density of the fermenter culture
to a value
2 6 of 2 (measured at 600 nm) by the addition of fresh, sterile fermenter
medium.
2 ~ The viscosity and viability of the formulation was stabilized by the
addition of a
2 s sterile suspension of Xanthan gum (prepared in distilled water) to the
diluted
2 9 fermenter culture until the final Xanthan concentration in the formulation
was 1
30 (w/v). The titre of Rhizobium meliloti strain NRG-34 in the liquid
formulation was
31 2 X 109 cells/ml.
22
216 0 7 8 Q PCTICA94100243
w O 94/25568
1 EXAMPLE 6
2 In 1990, a multi-year field research program was implemented to
3 compare the effects of a cold-tolerant Rhizobium meliloti strain (NRG-34)
with 2
4 temperate Rhizobium meliloti strains used in commercial inoculants (i.e.,
NRG-
185 and BALSAC) and uninoculated controls, on nodulation performance and
6 alfalfa production. Five research sites were established in the Peace River
region
of Alberta in the spring of 1990. All of the sites experienced at least some
time
at soil temperatures less than 10°C given their northern location. The
sites were
9 located in the County of Grande Prairie and were identified as follows
(legal
1 o description in brackets):
11 - Toews, West; (NW9-71-11-W6),
12 - Toews, East; (NE10-71-11-W6),
13 - Station Site #1; (Agriculture Canada Research Station;
14 NW36-71-10-W6), ,
- Station Site #2; (Agriculture Canada Research Station;
16 NE36-71-10-W6),
1~ - Hegland; (SW18-72-10-W6).
18 Four Rhizobium meiiloti inoculation treatments were compared: (a)
19 uninoculated control, (b) NRG-34, (c) NRG-185, (d) BALSAC. The treatment
2 o rows were 20 feet long and were arranged in a complete-randomized-block
21 design. Each treatment was replicated 6 times at each site. All sites were
22 sampled in the fall of 1990, spring 1991 and fall 1991. Nodule, shoot and
root
2 3 data were obtained from plant samples which were carefully excavated from
the
24 research sites. Hay production data was generated by harvesting each
treatment
2 5 row, determining the dry weight of hay produced/row, and then
extrapolating the
2 5 data to estimate hay productioNha. Strains were confirmed as cold-tolerant
if
2 ~ they exhibited improved nodulation and/or improved plant biomass
development
2 8 over the results from the temperate Rhizobium meliloti strains used in
2 9 commercial inoculants.
3 o Samples collected in the fall of 1990 indicated that alfalfa inoculated
31 with strain NRG-34, on average, showed improved nodulation in terms of
3 2 effective nodule numbers over alfalfa plants that were innoculated with
temperate
3 3 Rhizobium meliloti strains used in commercial inoculants and uninoculated
34 controls. NRG-34 inoculated alfalfa plants contained 12% more effective
nodules
23
'~: ~ fi~ 0'~ 8
WO 94/25568 PCT/CA94100243 -
1 than those inoculated with strain NRG-185. Nodulation
with the BALSAC strain
2 was equivalent to nodulation by strain NRG-34.
On average, alfalfa inoculated
3 with strain NRG-34 showed improved plant biomass
in terms of root and shoot
4 tissue production over alfalfa plants that were d with temperate
innoculate
Rhizobium meliloti strains used in commercial inoculantsand uninoculated
6 controls. The roots of alfalfa inoculated with ned 8% more
NRG-34 contai dry
7 matter than did roots inoculated with NRG-185, re dry matter
and 23% mo than
8 the BALSAC-treated plants. Shoot material productionlfa inoculated
by alfa with
NRG-34 was 16% greater than in the NRG-185-inoculatedplants, and
44%
1o greater than the plants inoculated with the BALSACThese results
strain. are
11 presented in Table 5.
12 Table 5: Comparison of Rhizobium meliloti strain
performance in
13 1990-seeded alfalfa field trials.
14
_______________________________________________________________________________
_____________________________
No. of Nodule Root Shoot
16 Test site Strain# nodules' dry wt~ dry wt3 dry wt4
17
_______________________________________________________________________________
___________---_______________
18 Toews, Control 0.63 0.67 0.452 0.659
19 West NRG-34 1.93 1.83 0.894 1.215
NRG-185 2.52 1.35 0.649 1.050
21 BALSAC 2.55 2.90 0.581 0.703
22 Toews, Control 3.93 0.78 0.890 1.290
23 East NRG-34 5.08 0.87 1.757 3.030
24 NRG-185 2.32 0.58 1.696 2.460
2 5 BALSAC 4.35 0.83 1.415 1.990
2 6 Station site Control 1.02 1.93 0.456 0.500
27 #1 NRG-34 3.83 2.87 0.858 1.062
28 NRG-185 1.05 4.08 0.927 1.270
2 9 BALSAC 1.47 2.97 0.802 0.997
3 0 Station site Control 0.45 0.67 0.509 0.545
31 #2 NRG-34 1.20 4.75 0.899 1.518
32 NRG-185 2.18 5.75 0.795 1.045
3 3 BALSAC 1.45 3.23 0.822 1.010
34 Hegland Control 1.57 3.48 0.509 0.545
35 NRG-34 7.83 2.25 0.610 0.785
3 6 NRG-185 9.65 2.75 0.574 0.734
3 7 BALSAC 10.23 2.42 0.476 0.574
24
O 94/25568 PCT/CA94/00243
1 Overall Control 1.52 1.53 0.562 0.704
2 average NRG-34 3.97 2.51 1.004 1.522
3 NRG-185 3.54 2.90 0.928 1.312
4 BALSAC 4.01 2.47 0.819 1.055
SED 0.76 0.61 0.086 0.134
______________________~________________________________________________________
___________________________
1. Number of nodules/plant.
s 2. Dry weights of nodules were determined by sampling the largest 24
nodules per root system, drying for 2 hours at 70°C and then recording
1 o dry weights. Data expressed as an average in grams of 24 nodules. If
11 less than 24 nodules were present on a root system, all nodules were
12 harvested, dried and weighed. Data expressed as average in grams of
13 total nodule count.
14 3. Dry weights of roots expressed as the average in grams of the total
number of plants present in a 20-cm2 sampling area.
16 4. Dry weights of shoots expressed as the average in grams of the total
1~ number of plants present in a 20-cm2 sampling area.
1s In the year of site establishment, alfalfa inoculated with NRG-34,
19 on average, produced 20% more hay than the uninoculated control treatment,
2 0 . and 10% more than the BALSAC treatment. By the end of the second year,
the
2 z total hay production by the NRG-34-inoculated alfalfa was 10% greater, on
22 average, than the uninoculated control treatment, and 6% greater than the
2 3 BALSAC treatment. The dry weight of hay produced by alfalfa inoculated
with
24 NRG-34 was equivalent to that produced by NRG-185-inoculated alfalfa. These
results are presented in Table 6.
WO 94/25568 PCT/CA94/00243
1 Table 6: Effects of
Rhizobium meliloti
strains on hay production
2 in 1990-seeded field trials*.
alfalfa
3 _________________________________________________________________
_
_ ______________________________________
___
Rhizobium
meliloti
strain
__
_________________________________________________________________________
6 Test site Control NRG-34 NRG-185 BALSAC
_______________________________________________________________________
_____________________________________
(a) 1990; Sept.
Toews, West 1307 1530 1486 1343
1 o Toews, East 2169 2451 2348 2225
11 Station site #1 911 1340 1311 1168
12 Station site #2 939 1225 1493 1189
13 Hegland 760 761 743 717
14 Mean (SED=84 ') 1217 1461 1474 1329
16 (b) 1991; June
1 ~ Toews, West 2505 2687 2423 2531
18 Toews,East 4928 4466 5055 4650
19 Station site #1 2500 3229 2962 2971
2 o Station site #2 2842 3356 3624 3115
21 Hegland 2450 2400 2646 2195
22 Mean (SED=108~~) 3045 3227 3342 3093
2 3 (c) 1991; Sept.
2 4 Toews, West 609 688 593 699
2 5 Toews, East 1360 1447 1377 1347
2 6 Station site #1 761 993 927 897
2 ~ Station site #2 799 824 859 698
2 8 Hegland 995 1004 1036 1012
2 9 Mean (SED=46 ~) 905 991 958 931
3 o Two -year total 25,835 28,401 28,883 26,757
31
_______________________________________________________________________________
_____________________________
3 2 * data expressed as
dry weight hay, kg/ha.
33 EXAMPLE 7
34 The effects of the cold-tolerant Rhizobium meliloti strain NRG-34
3 5 (ATCC # 55340) on nitrogen fixation and alfalfa crop quality were
determined by
3 6 total N analysis (LECO FP automated nitrogen analyzer, operating on the
Dumas
3 ~ principle of sample oxidation and measurement of liberated N) of the plant
3 8 tissues harvested in EXAMPLE 4, and then calculating total protein yield
based
26
w O 94/25568 2 ~ 6 ~ 7 8 0 PCT/CA94/00243
1 on total plant N content.
2 Data in Table 7 demonstrates that over two growing seasons, on
3 average, alfalfa inoculated with strain NRG-34 had surprisingly higher
nitrogen
4 content (i.e., % nitrogen) than the uninoculated control plants and plants
inoculated with the NRG-185 or Balsac strains.
6 Table 7: Effects of Rhizobium
meliloti strains on
nitrogen content
7 of forage1990-seededalfalfa field
trials*.
g
_______________________________________________________________________________
_____________________________
9 Rhizobium
meliloti
strain
__
_________________________________________________________________________
11 Test site Control NRG-34 NRG-185 BALSAC
12
_______________________________________________________________________________
_____________________________
13 (a) 1990; Sept.
14 Toews, West 2.21 2.35 3.32 2.38
Toews, East 2.40 2.20 2.29 2.26
16 Station site #1 2.44 2.42 2.35 2.35
17 Station site #2 2.66 2.63 2.56 2.69
1 s Hegland 2.70 2.72 2.65 2.72
19 Mean (SED=0.05"S) 2.48 2.46 2.44 2.48
2 0 (b) 1991: June
21 Toews, West 2.58 2.52 2.67 2.64
2 2 Toews, East 3.01 3.07 2.79 2.77
2 3 Station site #1 2.73 2.66 2.33 2.66
24 Station site #2 2.78 3.12 2.99 2.85
Hegland 2.96 3.03 2.89 2.90
2 6 Mean (SED=0.05**) 2.81 2.88 2.74 2.76
2 7 (c) 1991; Sept.
2 8 Toews, West 2.26 2.33 2.26 2.29
2 9 Toews, East 1.57 1.58 1.59 1.62
3 0 Station site #1 2.60 2.51 2.53 2.53
31 Station site #2 2.76 2.78 2.72 2.70
32 Hegland 2.35 2.71 2.43 2.32
3 3 Mean (SED=0.03**) 2.31 2.38 2.31 2.29
34
3 5 3-cut mean 2.53 2.57 2.49 2.51
3 6
_______________________________________________________________________________
_____________________________
37 * data expressed as % nitrogen.
27
~~so~.~a
WO 94/25568 PCTICA94/00243 -
1 Data in Table 8 demonstrates that over two growing seasons,
2 alfalfa inoculated with strain NRG-34 (ATCC # 55340) surprisingly produced
18
3 kg more total protein per hectare than plants inoculated with NRG-185, 80 kg
4 more total protein per hectare than plants inoculated with the BALSAC
strain,
and 98 kg more total protein per hectare than uninoculated control plants.
6 Table 8: Effects of Rhizobium meiiloti strains on protein yield
in 1990-seeded alfalfa field trials*.
g
_______________________________________________________________________________
_____________________________
9 Rhizobium meliloti strain
___________________________________________________________________________
11 Test site Control NRG-34 NRG-185 BALSAC
1 2
_______________________________________________________________________________
_____________________________
13 (a) 1990; Sept. 189 225 225 206
14 (b) 1991; June 535 581 572 534
(c) 1991: fall 131 147 138 133
16 2-year protein 855 953 935 873
1~ yield
18
_______________________________________________________________________________
_____________________________
19 * data expressed as kg protein/ha (calculated using mean hay production
2 o and nitrogen content data for each harvest date)
21 EXAMPLE 8
22 Rhizobium meliloti strain NRG-34 (ATCC # 55340) was used to
2 3 create 4 hybridoma cell lines using the method disclosed hereinabove,
within this
24 group, a hybridoma cell line designated as NRG-F12P92F, that produced a
2 5 distinct and unique monoclonal antibody designated as MAb-7. MAb-7 was
2 6 characterized using a commercial antibody typing kit and column gel
2~ chromatography, as an "Immunoglobulin micra" (IgM). The NRG-34 antigen to
2 8 which MAb-7 uniquely and specifically binds is a heat and periodate-stable
2 9 lipopolysaccharide. Extensive testing with 120 known strains of Rhizobium
3 o meliloti and Rhizobium trifolii confirmed conclusively that MAb-7 reacts
strongly
31 with strain NRG-34 (ATCC # 55340) and shows very little cross-reactivity
with
32 the other 119 strains (Figure 2).
28
~~ "~~ 78~
.~ O 94125568 PCTlCA94/00243
1 Figure 2 represents measurements of ELISA reactivity between
2 MAb-7 and the 120 strains tested (68 Rhizobium meliloti strains and 52
3 Rhizobium trifolii strains). Activities are expressed in terms of the
percentages
of the individual strain's reactivity (measured as absorbance) of that of the
s independent positive control. The positive control value is defined as 100%
and
represents the reactivity between MAb-7 and the strain (i.e., NRG-34) used to
immunize the mouse from which MAb-7 was derived.
8 EXAMPLE 9
9 Alfalfa seed was inoculated with Rhizobium melilotistrains NRG-34,
Zo NRG-185and BALSAC, then planted in the spring of 1989 at a field site in
the
1i Peace River region of Alberta. Uninoculated alfalfa seed was planted as the
12 control treatment. Plants were carefully excavated in the fall of 1990, and
the
13 spring and fall of 1991, the root systems washed, nodules removed and
stored
14 frozen for processing at a later date. A modified ELISA assay as disclosed
hereinabove, using MAb-7 for detection of NRG-34, and a polyclonal antibody
15 for detection of NRG-185, was performed on the nodules to determine the
1~ nodule occupancy rate by these two strains.
18 The results of the assays indicate that, compared to the temperate
19 commercial inoculant strain NRG-185, strain NRG-34 is an extremely
aggressive
2 o and efficient competitor for nodule occupancy. At the end of the first
growing
2z season, NRG-34 comprised 80.6% of nodule rhizobia in alfalfa inoculated
with
22 this strain, while NRG-185 was resident in only 50% of the nodules
collected
23 from plants inoculated with NRG-185 (Table 7). In the spring of the
following
24 year, the % nodule occupancy by NRG-185 was only 25% and dropped further
to less than 12% by the end of the second growing season Table 7). In
contrast,
2 6 the % nodule occupancy by NRG-34 in the spring of 1990 was 60% and by the
2 ~ end of the second growing season, the % nodule occupancy was more than
28 double that of the commercial inoculant strain NRG-185 Table 7). These
results
29 indicate that NRG-34 is a surprisingly superior nodule resident and will
have
3 o extended benefits to alfalfa production as compared to NRG-185.
29
CA 02160780 1998-09-21
1 Table 9: Percent nodule occupancy by strains
2 NRG-34 and NRG-185 on alfalfa inocu lated
3 in 1989 with 3 strains of R. meliloti.
4
_______________________________________________________________________________
____________________
% nodule occupancy
_____________________________ ________________________
7 Inoculant NRG-34 NRG-185
g
_______________________________________________________________________________
_____________________
9 (a) September 1989
Control 0.7 23.6
11 N RG-34 80.6 5.6
12 N RG-185 1.4 50.7
13 (b) June 1990
14 Control 0 8.3
N RG-34 60.4 2.8
16 NRG-185 0 25.0
1~ (c) September 1990
18 Control 0 0
19 NRG-34 22.4 2.4
NRG-185 0 11.9
2 1
_______________________________________________________________________________
________________________
22 All publications mentioned in this specification are indicative of the
23 level of skill of those skilled in the art to which this invention
pertains.
24 Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of understanding,
it will
2 6 be obvious that certain changes and modifications may be practised within
the
2 ~ scope of the appended claims.
