CA2160780C - Cold-tolerant strains of rhizobium species - Google Patents

Abstract

A process is disclosed for the selection and characterization of cold-tolerant strains of Rhizobium species, and the subsequent use of these cold-tolerant strains in inoculant compositions to improve nodulation nitrogen fixation and biomass development of legume forage crops. The selected cold-tolerant Rhizobium strains are employed as immunogens for the creation of hybridoma cell lines that produce monoclonal antibodies specific to the immunogen strains. The monoclonal antibodies enable detection of the immunogen cold-tolerant Rhizobium meliloti strains in cultures, nodules, inoculant compositions and soils.

Description

O 94/25568 216 0'~ 8 0 pCT/CA94100243 1 "COLD-TOLERANT STRAINS OF RHIZOBIUM SPECIES' 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.

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 __. 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 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:

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 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 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 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-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.

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

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.

.-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 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.

.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 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.

:.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 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

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.

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) 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.

2 2 Doubling time (h) No. of strains 2 4 Less than 20 1 2 ~ Greater than 50 79 2 s Total 97 2~ ss 7ss 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 ).

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 1 in each growth pouch were inoculated with 1-ml aliquots (1X108 cells/ml) of 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.

., O 94/25568 ~ PCTICA94/00243 1 Table 2: Number of effective nodules formed by Rhizobium 2 meliloti strains at cold root temperatures.

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.

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.

Root Temperature (C) 6 __________________________________ ________________~______-Strain # 9 10 11 12 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 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 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 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 cells/gram.

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.

w O 94/25568 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 '~: ~ fi~ 0'~ 8 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.

_______________________________________________________________________________ _____________________________ No. of Nodule Root Shoot 16 Test site Strain# nodules' dry wt~ dry wt3 dry wt4 _______________________________________________________________________________ ___________---_______________ 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 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.

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 _______________________________________________________________________________ _____________________________ 3 2 * data expressed as dry weight hay, kg/ha.

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 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

_______________________________________________________________________________ _____________________________ 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 3 5 3-cut mean 2.53 2.57 2.49 2.51 _______________________________________________________________________________ _____________________________ 37 * data expressed as % nitrogen.

~~so~.~a 1 Data in Table 8 demonstrates that over two growing seasons, 2 alfalfa inoculated with strain NRG-34 (ATCC # 55340) surprisingly produced 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

_______________________________________________________________________________ _____________________________ 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 _______________________________________________________________________________ _____________________________ 19 * data expressed as kg protein/ha (calculated using mean hay production 2 o and nitrogen content data for each harvest date) 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).

~~ "~~ 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.

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.

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.

_______________________________________________________________________________ ____________________ % 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 _______________________________________________________________________________ ________________________ 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.

Claims (27)

What is claimed is:

1. A method for screening a culture collection of one or more test strains of Rhizobium species to detect and select cold-tolerant strains for a legume crop group, comprising:
(a) individually culturing the test Rhizobium strains at a temperature of less than about 10°C;
(b) selecting those Rhizobium strains from step (a) which exhibit growth;
(c) inoculating test legume seeds of the legume crop group with the selected strains from step (b);
(d) germinating the inoculated seeds and growing test legume plants under controlled conditions where the temperature is maintained at between about 9 to 1 5°C for a period of time sufficient for nodules to be established on the legume plants, (e) selecting those Rhizobium strains from step (d) for which the test legume plants exhibit nodulation;
(f) inoculating test legume seedlings of the legume crop group with the selected strains from step (e) and inoculating control legume seedlings of the legume crop group with one or more temperate Rhizobium strains;
(g) growing the test and control legume seedlings under controlled conditions in which the roots are maintained at a temperature of less than about 1 0°C while the shoot temperature is maintained at greater than about 15°C, for a time sufficient for the control plants to establish effective nodules; and (h) selecting, as cold-tolerant Rhizobium strains for the legume crop group, those Rhizobium strains from step (g) for which the test legume plants exhibit improved nodulation over that exhibited by the control legume plants.

2. The method according to claim 1, wherein strains of the following Rhizobium species are screened with the following legume crop groups:
Rhizobium species screened Legume Crop Group Rhizobium meliloti alfalfa or sweet clover Rhizobium leguminosarum peas or lentils Rhizobium phaesolii beans Bradyrhizobium japonicum soybeans Rhizobium trifolii red clover

3. The method according to claim 1, wherein strains of Rhizobium meliloti are screened with alfalfa or sweet clover as the legume crop group.

4. The method according to claim 2 wherein in step (b), the selected strains are screened with a polyclonal antibody for that Rhizobium species to select only biologically pure cultures of that Rhizobium species.

5. The method according to claim 2 wherein in step (f), the control legume plants are inoculated with one or more temperate Rhizobium meliloti strains selected from a group consisting of 102F34, BALSAC, URB-165 and NRG-1 85.

6. The method according to claim 5 wherein in step (9) the ambient shoot temperature is maintained at about 20 to 30°C.

7. The method according to claim 6 wherein strains of Rhizobium meliloti are screened and wherein, in steps (c) and (f), the test and control legume seeds or seedlings are alfalfa or sweet clover.

8. The method according to claim 2 which includes the further steps of:
(i) inoculating second test legume seeds of the legume crop group with the selected strains from step (h) and inoculating second control legume seeds of the legume crop group with one or more temperate strains of Rhizobium species;
a) growing legume field crops from the inoculated test and control legume seeds; and (k) confirming as cold-tolerant Rhizobium strains, those strains from step a) for which the test field crop exhibits improved nodulation or plant biomass development over that obtained for the control field crop.

9. The method according to claim 8 wherein in step (i), the control legume seeds are inoculated with one or more temperate Rhizobium meliloti strains selected from a group consisting of 102F34, BALSAC, URB-165 and NRG-1 85.

10. The method according to claim 9 wherein in step (j), the legume field crop is grown in field conditions which include some time at soil temperature of less than about 10°C.

11. The method according to claim 11 wherein strains of Rhizobium meliloti are screened and wherein the legume crop group is alfalfa or sweet clover.

12. A biologically pure culture of a cold-tolerant strain of Rhizobium species, whenever obtained by the method of claim 1.

13. A biologically pure culture of a cold-tolerant strain of Rhizobium species selected from R. meliloti, R. leguminosarum, R. phaesolii, Bradyrhizobium japonicum and R. trifolii, whenever obtained by the method of claim 2.

14. A biologically pure culture of a cold tolerant strain of Rhizobium meliloti, whenever obtained by the method of claim 3.

15. A biologically pure culture of cold-tolerant Rhizobium meliloti strain NRG-34 (ATCC # 55340).

16. A method to promote growth of a legume crop group comprising inoculating legume seeds of the legume crop group with agriculturally effective amounts of a cold-tolerant strain of Rhizobium species of claim 13, wherein the Rhizobium species and legume crop groups are matched as follows:

Rhizobium species screened Legume Crop Rhizobium meliloti alfalfa or sweet clover Rhizobium leguminosarum peas or lentils Rhizobium phaesolii beans Bradyrhizobium japonicum soybeans Rhizobium trifolii red clover

17. The method according to claim 16, wherein the cold-tolerant strain is Rhizobium meliloti NRG-34 (ATCC #55340) and the legume crop group is alfalfa or sweet clover.

18. An agricultural inoculant composition comprising a carrier medium in admixture with a cold-tolerant strain of Rhizobium species of claim 12.

19. The composition according to claim 18, wherein the cold-tolerant strain of Rhizobium is of claim 13.

20. The composition according to claim 18, wherein the cold- tolerant strain is Rhizobium meliloti NRG-34 (ATCC # 55340).

21. The composition according to claim 18 wherein the form of the carrier medium is selected from a group consisting of liquids, powders, granulesand gels.

22. A monoclonal antibody whenever produced using as immunogens, the cold-tolerant strains of Rhizobium meliloti of claim 14.

23. The monoclonal antibody of claim 22, MAb-7, produced using Rhizobium meliloti strain NRG-34 (ATCC No. 55340) as the immunogen and NRG-F12P92F as the hybridoma cell line.

24. A method for detecting or quantifying cold-tolerant Rhizobium meliloti strains in test samples of culture media, nodule tissue, inoculant compositions and soil, comprising assaying the test sample with the monoclonal antibodies of claim 22.

25. The method according to claim 24 wherein the cold-tolerant strain is Rhizobium meliloti strain NRG-34 (ATCC No. 55340).

26. The method according to claim 25 wherein the assay is an ELISA
assay.

27. The method according to claim 25 wherein the assay is an immunoblot assay.