Rhizobium huakuii sp. nov. Isolated from the Root Nodules of As t raga ...

INTERNATIONAL JOURNALOF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 275-280 0020-7713/91/020275-06$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Vol. 41, No. 2

Rhizobium huakuii sp. nov. Isolated from the Root Nodules of As traga 1us sin icus W. X. CHEN,* G. S. LI, Y. L. QI, E. T. WANG, H . L. YUAN, AND J. L. LI Department of Microbiology, College of Biology, Beijing Agricultural University, Beijing, People’s Republic of China Nine bacterial strains isolated from root nodules of Astragalus sinicus were compared with 41 reference strains, including the type strains of the type species of the genera Rhizobium, Bradyrhizobium, and Agrobacterium, by performing a numerical analysis of 200 phenotype features. Representative strains belonging to different clusters were further compared with similar bacteria by using data from gel electrophoresis of whole-cell proteins, DNA G C content data, and DNA-DNA hybridization data. The rhizobial strains isolated from nodules of A . sinicus constitute a distinct homology group that is quite different from previously described Rhizobium, Bradyrhizobium, and Agrobacterium species and from strains isolated from other Astragalus species. We propose the name Rhizobium huakuii sp. nov. for the strains isolated from A . sinicus. Type strain CCBAU 2609 (= 103) has been deposited in the Culture Collection of Beijing Agricultural University, Beijing, People’s Republic of China.

+

Bacteria which form nitrogen-fixing nodules on leguminous plants are currently divided into the following four genera: Rhizobium, Bradyrhizobium, Azorhizobium, and Sinorhizobium (2, 5, 9). The genus Rhizobium comprises three species, Rhizobium Eeguminosarum, Rhizobium meliloti, and Rhizobium loti. The genus Bradyrhizobium includes one well-defined species, Bradyrhizobium japonicum. The genus Azorhizobium contains only one named species, Azorhizobium caulinodans. The genus Sinorhizobium consists of two species, Sinorhizobium fredii and Sinorhizobium xinjiangensis. Astragalus sinicus is an important winter-growing green manure in the southern part of the People’s Republic of China. Rhizobia have been found in the nodules of this legume only in the People’s Republic of China and Japan. Selected strains of these bacteria have been used as an inoculant which has been widely applied in the southern part of the People’s Republic of China for the last 30 years with good results; usually, this inoculant increases host plant yields by 15 to 30% or more and increases the nitrogen content in the plants by 2.0 to 2.5% compared with uninoculated plants. These bacteria contain up to four plasmids that are 10 to 300 kb. long. Thefix gene and the nod gene might be on these plasmids (24, 28). Utilization of respiratory substrates by these bacteria has been investigated with a Warburg respirometer; sucrose was not utilized, glucose and fructose were utilized at a low rate, and succinate was the most effective substrate (13). In order to determine the taxonomic position of these bacteria, we analyzed strains isolated from As tragalus sinicus collected from different areas of the People’s Republic of China and compared them with isolates obtained from other Astragalus species and with representatives of the genera Rhizobium, Bradyrhizobium, Agrobacterium, and Sinorhizobium by performing a numerical analysis of phenotypic features, a gel electrophoresis analysis of the whole-cell proteins, a DNA base composition analysis, and a DNADNA hybridization analysis. The infective properties of the rhizobia obtained from Astragalus sinicus also were examined.

* Corresponding author.

Our results led us to propose the name Rhizobium huakuii sp. nov. for the strains isolated from Astragalus sinicus. The type strain is strain CCBAU 2609. MATERIALS AND METHODS Bacterial strains. A total of 58 strains were used in this study (Table 1). Ten strains were isolated from Astragalus sinicus, seven strains were isolated from other leguminous plants, and the other strains were representatives of the genera Rhizobium, Bradyrhizobium, Agrobacterium, and Sinorhizobium . Phenotypic features and methods of analysis. The strains were characterized by studying the following features: utilization of organic substrates as sole carbon and nitrogen sources; requirement for vitamins; tolerance to dyes, antibiotics, and sodium chloride; pH range for growth; reaction in litmus milk; reduction of methyl blue, nile blue, and nitrate; growth in peptone broth; growth at 37 and 39°C; flagellum type; and other characteristics. Most of the biochemical tests were conducted by using plates inoculated with a multipoint inoculator (lo), using about lo5 cells per point as the inoculum. Other tests were conducted in broth. Unless otherwise indicated, the cultures were incubated at 28°C. The growth of fast- and slowgrowing strains was observed after incubation for 3 to 7 and 7 to 10 days, respectively. Carbohydrate utilization and organic acid utilization were determined on the medium of White (26), which contained the trace elements of medium Cs7 (16). Sole nitrogen source utilization was examined by using the same medium, except that NaNO, was replaced with various nitrogen sources at a concentration of 0.1% (wthol). The requirements for vitamins and growth factors were investigated by using the medium of Zhou and Cao (27). Tolerance to antibiotics, dyes, and NaCl at various concentrations and the pH range for growth were examined by using YMA (9) and the method of Thompson and Skerman (22). Nitrate reduction was tested by using a modification of the method of Pohlman (17), as described previously (2). Flagella were observed by using the modified Fontana staining method (19). Computer analysis. Coding of traits, calculating similarity values by using the Ssm formula, determining the center

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TABLE 1. Origins and hosts of the bacterial strains examined Strain

R. meliloti strains 102F28 HI USDA 1002T' R. leguminosarum biovar trifolii strains TA1 8101 T47 162x68 R. leguminosarum biovar viceae strains 1-2 1-3 03-3 s54 128 C53 USDA 2370T R. leguminosarum biovar phaseoli strains 127 K17 F48 S . fredii strains 440 6-1 131 Rhizobium sp. strains T A106 S52 Bs-12 PL-52 B23-106-26 B19-40 38 7034 103T (= CCBAU 2609)T B. japonicum strains b, 122-3 305 B15 2135 2110 Y11 USDA 6* Bradyrhizobium sp. strains 23-4 4 4-1 7-1 5-1 C 09-23 1-6 Tha 201 1035 351 Agrobacterium tumefaciens strains B6S3 C58 Agrobacterium radiobacter 1.150 Rhizobium sp. strains CB81 380 CA8561

Host

Origin"

Source'

Heilongjiang United States

UCD CCBAU USDA

Australia Guangxi Hubei United States

IOC (CAAS) SFRI (CAAS) HAU USDA

Vicia sepium Vicia sepium Vicia faba Vicia villosa

Beijing Beijing Xinjiang Hubei United States United States

CCBAU CCBAU CCBAU HAU USDA USDA

? ?

United States Argentina

USDA SFRI (CAAS)

Glycine soja Vigna sp. Glycine soja

Shanghai Beijing Hebei

CCBAU CCBAU CCBAU

Astragalus adsurgens Astragalus sinicus A s traga lus sin icu s Astragalus sinicus Astragalus sinicus Astragalus sinicus Astragalus sinicus Astvagalus sinicus Astragalus sinicus Astragalus sinicus

Heilongjiang Hubei Hubei Hubei Hubei Hubei Hubei Nanjing Nanjing Nanjing

CCBAU HAU HAU HAU HAU HAU HAU NAU NAU NAU

Glycine soja Glycine soja Glycine soja Glycine max Glycine max Glycine max Glycine soja Glycine max

Heilongjiang Hubei Hubei Liaoning United States United States Hubei United States

CCBAU IOC (CAAS) IOC (CAAS) IOC (CAAS) SFRI (CAAS) SFRI (CAAS) SFRI (CAAS) USDA

Desomodium canum Stylosanthes gracilis Stylosanthes gracilis Stylosanthes gracilis Stylosanthes gracilis Kummerowia strita Desomodium canum Macrotyloma aillare Arachis hypogaea Aruchis hypogaea Erythrina sp.

Xishuangbanna Guangxi Guangxi Guangxi Guangxi Heilongjiang Xinjiang Guangxi United States Guangxi Guangdong

IOC (CAAS) SFRI (CAAS) SFRI (CAAS) SFRI (CAAS) SFRI (CAAS) CCBAU CCBAU SFRI (CAAS) SFRI (CAAS) SFRI (CAAS) CCBAU

Medicago sativa Melilotus albus ?

Trifolium repens Trifolium repens Trifolium subterraneum 3

? ?

CCBAU CCBAU CCBAU Leucaena leucocephala Leucaena leucocephala Astragulus membranaceus

Australia Hainan Beijing

CCBAU CCBAU SFRI (CAAS) Continued on following pagc

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277

TABLE 1-Continued Strain

Host

CA8593 A16 CA8213

Astragalus Astragalus Astragalus Astragalus Astragalus

K T2

membranaceus adsurgens adsurgens aliginosus sinicus

Origin

Beijing Beiji ng Beijing Heilongj iang N anjing

Source’

SFRI ( C A A S ) SFRI ( C A A S ) SFRI ( C A A S ) CCBAU NAU

All localities within the People’s Republic of China unless indicated otherwise.

’CCBAU, Culture Collection of Beijing Agricultural University, Beijing, People’s Republic of China; HAU, Huazhong Agricultural University, Wuhan,

a

People’s Republic of China; IOC (CAAS), Institute of Oil Crops, Chinese Academy of Agricultural Science, Wuhan, People’s Republic of China; NAU, Nanjing Agricultural University, Nanjing, People’s Republic of China; SFRI (CAAS), Soil and Fertilizer Research Institute, Chinese Academy of Agricultural Science, Beijing, People’s Republic of China; UCD, University of California, Davis; USDA, United States Department of Agriculture, Beltsville, Md. ‘. T = type strain.

strains of the groups, and clustering by single linkage were all performed by using the methods of Sneath and Sokal(21). Polyacrylamide gel electrophoresis. Protein samples were prepared from whole cells of each strain grown in YMB (9) for 1 day (fast growers) or 2 days (slow growers). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by using the method of Laemmli (12). DNA base composition. DNA was prepared by the method of Johnson (8). The average G + C content of the DNA of each strain was measured by using the thermal denaturation method (15) and the equation of De Ley (3). Escherichia coli K-12 was used as the standard. DNA-DNA hybridization. DNA samples were treated by using the method of De Ley et al. (4), and the levels of homology were determined by using the initial renaturation rate method (13). Infective properties. Three strains isolated from Astragalus sinicus were examined for their symbiotic relationships with other leguminous plants. Inoculation tests were carried out by using the methods of Vincent (23) and a chamber kept at 28°C during the day and 20°C during the night with illumination of 15,000 lx for 14 h/day. Smallseeded legumes were grown in sterilized vermiculite in glass tubes (200 by 30 mm) plugged with cotton wool; these plants were completely enclosed. Large-seeded legumes were grown in jars by using the method of Vincent (23), with some modification. The nutrient solution used for the plants contained 0.03 g of Ca(NO,),, 0.46 g of CaSO,, 0.14 g of KZHPO,, 0.07 g of KCl, 0.06 g of MgSO, 7H20, 0.07 g of iron citrate, 1ml of a trace element solution, and 1,000 ml of distilled water. The trace element solution contained 2.86 g of H,BO,, 1.8 g of MnSO,, 0.2 g of ZnSO,, 0.8 g of CuSO, . 5H20, 0.02 g of HMoO,, and 1,000 ml of distilled water. Inoculation was accomplished by placing the germinating seeds in a bacterial cell suspension containing about lo8 cells per ml; 1 or 2 ml of this suspension was added to each container around the seedling after sowing. Plants were grown for 3 to 4 weeks. The nitrogenase activity of nodules was tested by using the ethylene reduction method (20). *

RESULTS AND DISCUSSION The first 50 strains listed in Table 1 were compared by using numerical taxonomy. These 50 strains were divided into four phenotypic groups (Fig. l ) , corresponding to the genera Rhizobium, Agrobacterium, Sinorhizobium, and Bradyrhizobium. We found that all nine isolates from Astragalus sinicus were closely related, clustering as one phenon within the genus Rhizobium, and were clearly separated from R . meliloti and R . leguminosarum and from strain T isolated from Astragalus adsurgens. Similar results were

obtained by Chen et al. (2), who used the average linkage method of numerical taxonomy. The phenotypic features of this phenon are given below, and the differential characteristics of the phenon (isolates from Astragalus sinicus), R. meliloti, and R. leguminosarum are shown in Table 2. The unique morphological feature is that strains of R. meliloti and R . leguminosarum are peritrichow, but strains isolated from Astragalus sinicus have a single polar or subpolar flagellum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and UV scanning were used to determine the patterns of whole-cell proteins. Seven isolates from Astragalus sinicus had almost identical polyacrylamide gel electrophoresis patterns, which were quite different from the patterns of R . meliloti, R . leguminosarum, S . fredii, B. japonicum, and Agrobacterium tumefaciens strains. These protein patterns also indicated that the isolates obtained from Astragalus sinicus constitute a homologous group that can be distinguished from previously described rhizobial species. The contents G + C of six representative Rhizobium strains were 60 to 61 mol%, the G + C contents of six Bradyrhizobium strains were 63 to 65 mol%, and the G + C contents of seven isolates from Astragalus sinicus were 59 to 64 mol% (in the range of values for Rhizobium spp. [9]). The G+C contents of isolates from other Astragalus species were also within the range of values for Rhizobium species. DNA hybridization was carried out with seven strains obtained from Astragalus sinicus, representative strains isolated from Astragalus membranaceus, Astragalus adsurgens, and Astragalus aliginosus, and Rhizobium and Bradyrhizobium strains. All of the strains isolated from Astragalus sinicus which we examined exhibited high levels of relateness to each other (Table 3) and low levels of relateness to the reference strains examined (Table 4). Three strains isolated from Astragalus sinicus were use to test cross-infection in 16 leguminous species. Nodulation occurred in three of five Vicia villosa test plants, four of seven Phaseolus vulgaris test plants, four of five Sesbania sp. test plants, four of four Astragalus aliginosus test plants, and four of six Astragalus adsurgens test plants. Nitrogenase activity was demonstrated in infected plants of Vicia villosa, Phaseolus vulgaris, and Sesbania sp. We found no nodules on six Astragalus membranacens plants, six Astragalus mongholicus plants, four Hedysarum mongolicum plants, nine Pisum sepium plants, three Trifolium repens plants, six Medicago sativa plants, six Melilotus albus plants, five Lotus corniculatus plants, nine Glycine max plants, six Lupinus sp. plants, and nine Vigna sinensis plants. Astragalus sinicus formed nodules only with bacteria


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The level of Similarity

Strain Number

iE 38

,

7034 103 Bs-12 819-40 B23-106-28

R, huakui i

A106

-

s52 PL-52 8101

Rhizobi um R, le~uminosatum

USDA 2370T F48

127 K17 s5 1 128C53

T

-

USDA 1002

1 -+~

E':

muibozihroniS,-]

6-1 4

~

1

1

5-1 4-1 1-1 09-23 1-6 122-3 3 05 B15 bl 2110 Y11

- Bradyrhizobium

USDA 6 T 35 1

FIG. 1. Dendrogram.

isolated from Astragalus sinicus and with 1 of 30 other strains of bacteria isolated from various leguminous plants (1).Bacteria isolated from Astragalus sinicus and Astragalus adsurgens would not cross-infect the two plant species (14). On the basis of the results of numerical taxonomy, polyacrylamide gel electrophoresis, DNA base composition, DNA-DNA hybridization, and cross-inoculation analyses, we propose that the rhizobia isolated from Astragalus sinicus should be assigned to a new species, Rhizohium huakuii (hua.ku'i.i. N.L. gen. n. huakuii, in honor of the Chinese professor of soil microbiology Huakui Chen, who studied

rhizobia isolated from Astragalus sinicus for nearly 50 years). Description of Rhizobiurn huakuii sp. nov. Rods (0.5 to 0.9 by 1.2 to 3.0 pm). Gram negative. Nonsporeforming. Aerobic, possessing a respiratory type of metabolism with oxygen as the terminal electron acceptor. Motile by means of a single polar or subpolar flagellum. Cells usually contain granules of poly-P-hydroxybutyrate. Colonies are circular, convex, semitranslucent, raised, and mucilaginous; they are usually 2 to 4 mm in diameter within 5 or 6 days on YMA. Chemoorganotrophic, utilizing a wide range of carbohy-


VOL.41, 1991

RHIZOBIUM HUAKUII SP. NOV.

TABLE 2. Differential characteristics of R . huakuii, R . legurninosarurn, and R . rneliloti Characteristic

~

~

-

+ +

+

A106

103T

0 4 0 38 28 27 0 0 0 32 50 53

0 7 0 40 27 35 2 0 0 30 52 62

0 13 0 26 19 25 0 2 11 13 52 64

~

~

~~~~

The DNA homology values are the means of the values from two or three replications. The calculated relative error was less than 3.5%. The names in parentheses are the host plants of the strains. 'I

~

~

~

~

~

less than 95% but more than 5% of the strains are positive; -, less than 5% of the strains are positive.

'

_

_

_

~

C,

drates, organic acids, and amino acids as sole carbon sources. Arabinose, cellobiose, fructose, fumarate, glucose, lactose, malate, maltose, raffinose, rhamnose, sodium lactate, sodium oxalate, succinate, and sucrose are utilized. Alanine, ammonium tartrate, asparagine, caffine, cellulose, cysteine, dulcito!, ferulic acid, fucose, p-hydroxybenzoic acid, inositol, melibiose, pyrocatechol, salicyto!, sodium acetate, sodium benzonate, sodium citrate, sodium formate, sodium hippurate, sodium salicylate, sorbose, starch, and vanillic acid are not utilized. Produces an acid reaction which is indicated by brommethyl blue in mineral salts medium containing mannitol. Ammonium nitrate, urea, and many amino acids, including arginine, citrulline, lysine, methionine, ornithine monohydrochloride, threonine, tryptophan, and tyrosine, are utilized as sole nitrogen sources. DL-Leucine, D-methionhe, and valine are not utilized. Peptone is poorly utilized. Some strains require thiamine, nicotinamide, and riboflavin for growth. The optimum growth temperature is 25 to 30°C; most strains grow at 37°C but not at 39°C. The pH range for growth is 5.0 to 9.5; the optimum pH is 6 to 8. Methyl blue is reduced. Nitrate and nitrite are not reduced. Produces a serum zone in litmus milk. The G + C content of the DNA is 59 to 64 mol%, as determined by the thermal denaturation method. TABLE 3. Levels of DNA homology between isolates from Astrugalus sinicusa

PL-52 A106 T2 103T S52 B23-106-26 38

PL-52

CA8561 (Astragalus rnernbranacens)b A16 (Astragalus adsurgens) K (Astragalus uliginosus) R . leguminosarurn USDA 2370T R . rneliloti USDA 1002T R . loti ATCC 33669T Bradyrhizobium sp. strain USDA 3045 B . japonicum USDA 110 B . japonicurn USDA 6T Agrobacteriurn turnefaciens C5* CB81 (Leucaena leucocephala) 3 8 0 (Leucaena leucocephala)

+ +

t

% Homology with strain:

Strain

+ + + +

" +, At least 95% of the strains are positive;

Strain

TABLE 4. Levels of DNA relatedness among strains of R . huakuii, strains isolated from other leguminous plants, and type strains of Rhizobium, Bradyrhizobiurn, and Agrobacterium species"

R . huakuii R . leguminosarum R . meliloti

Utilization of Sodium lactate DL-Tyrosine L-Cysteine DL-Tryptophan DL-Threonine Tolerance to: Polymyxin (10 bg/ml) Paromomycin (30 pg/ml) Reddish orange (0.01%) Fuchsin basic (0.1%) Growth at: pH 4.9 pH 9.5 39°C Growth in peptone broth Polar and subpolar flagella Peritrichous Reduction of methylene blue

279

% Homology with strain:

PL-52

A106

T,

103T

S52

B23-106-26

38

100

83 100

74 96 100

73 98 101 100

78 86 85 86 100

78 81 73 75 81 100

79 99 92 101 85 79 100

" The DNA homology values are the means of the values from three or four replications. The relative error was less than 4.1%.

Additional characteristics are shown in Table 2. The type strain of Rhizobium huakuii is strain 103 (= CCBAU 2609), which is the center strain of this species group. This strain conforms to the description given above for the species. In addition, it uses galactose, L-glutamate, D-mannose, sodium gluconate, sodium pyruvate, sorbitol, and trehalose as sole carbon sources and DL-asparagine and DL-serine as sole nitrogen sources. It requires nicotinamide and riboflavin for growth and grows on YMA containing 1% NaCl. It has been deposited in the Culture Collection of Beijing Agricultural University, Beijing, People's Republic of China. ACKNOWLEDGMENTS

We are particularly indebted to Y . F. Zhao and J. C. Ma (Institute of Microbiology, Chinese Academy of Science, Beijing, People's Republic of China), who did the computer analyses. Funding for this research was provided by the National Natural Sciences Foundation and the National Educational Committee Foundation of the People's Republic of China. REFERENCES 1. Chen, H. K., and M. K. Shu. 1944. Note on the root-nodule bacteria of Astragalus sinicus. Soil Sci. 58:291-293. 2. Chen, W. X., G. H. Yan, and J. L. Li. 1988. Numerical taxonomic study of fast-growing soybean rhizobia and a proposal that Rhizohiurn fredii be assigned to Sinorhizobiurn gen. nov. Int. J. Syst. Bacteriol. 38:392-397. 3. De Ley, J. 1970. Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J. Bacteriol. 101:738-754. 4. De Ley, J., H. Cattoir, and A. Reynauts. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12:133-142. 5. Dreyfus, B., J. L. Garcia, and M. Gillis. 1988. Characterization of Azorhizobiurn caulinodans gen. nov., sp. nov., a stemnodulating nitrogen-fixing bacterium isolated from Sesbania rosrrate. Int. J. Syst. Bacteriol. 38539-98. 6. Graham, P. H. 1964. The application of computer technique to the taxonomy of the root-nodule bacteria of legumes. J. Gen. Microbiol. 35511-517. 7. Graham, P. H., and C. A. Parker. 1964. Diagnostic features in the characterisation of the root-nodule bacteria of legumes. Plant Soil 20:383-396. 8 . Johnson, J. L. 1985. Determination of DNA base composition,


280

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INT. J . SYST.BACTERIOL.

DNA reassociation and DNA hybridization of bacterial nucleic acid, p. 1-74. In G. Gottschalk (ed.), Methods in microbiology, vol. 18. Academic Press, Inc. (London), Ltd., London. 9. Jordan, D. C. 1984. Rhizobiaceae, p. 234-256. In N. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 10. Josey, D. P., J. L. Beyhon, A. W. B. Johnson, and J. E. Beringer. 1979. Strain identification in Rhizobium using intrinsic antibiotic resistance. J. Appl. Bacteriol. 46:343-350. 11. Kersters, K. 1985. Numerical methods in the classification of bacteria by protein electrophoresis, p. 337-368. In M. Goodfellow, D. Jones, and F. G. Priest (ed.), Computer-assisted bacterial systematics. Academic Press, Inc. (London), Ltd., London. 12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 13. Leu, W. J., and C. S. Fan. 1985. Study of energy sources of the symbiotic nitrogen fixation by bacteroids of As. sinicus. J. Nanjing Agric. Univ. 1:40-46. (In Chinese.) 14. Ning, G. Z. 1988. Infective properties of strain from Astrugalus adsurgens to Astragalus sinicus. J. Microbiol. 856. (In Chinese.) 15. Marmur, J., and P. Doty. 1962. Determination of the base composition of DNA from its thermal denaturation temperature. J. Mol. Biol. 5109-118. 16. Pagan, J. D., J. J. Child, W. R. Scowcroft, and A. H. Gibson. 1975. Nitrogen fixation by Rhizobium cultured on a defined medium. Nature (London) 256:406-407. 17. Pohlman, G. C. 1931. Changes produced in nitrogenous compounds by Rhizobium meliloti and R . japonicum. Soil Sci. 31:385. 18. Seidler, R. J., and M. Mandel. 1971. Quantitative aspects of

19. 20.

21. 22. 23. 24. 2.5. 26. 27. 28.

DNA renaturation, base composition, state of chromosomal replication, and polynucleotide homologies. J. Bacteriol. 106: 608-614. Skerman, V. B. D. 1967. A guide to the identification of the genera of bacteria, 2nd ed. The Williams & Wilkins Co., Baltimore. Slonger, C. 1969. Symbiotic effectiveness and N, fixation in nodulated soybean. Plant Physiol. 44:160&1668. Sneath, P. H. A., and R. B. Sokal. 1973. Numerical taxonomy. The principles and practices of numerical classification. W. H. Freeman and Co., San Francisco. Thompson, J. P., and V. B. D. Skerman. 1979. Azotobacteriaceae. Academic Press, Inc., New York. Vincent, J. M. 1970. A manual for the practical study of the root-nodule bacteria. Blackwell Scientific Publications, Ltd., Oxford. Wang, C. L., and J. B. Chen. 1988. The plasmid patterns and antibiotic resistance of the rhizobium strains of Astragalus sinicus. J. Huazhong Agric. Univ. 7:lS-21. (In Chinese.) Wetmer, J. G. 1976. Hybridization and renaturation kinetics of nucleic acids. Annu. Rev. Biophys. Bioeng. 5337-361. White, L. 0. 1972. The taxonomy of the crown gall organism Agrobacterium tumefaciens and its relationship to rhizobia and other agrobacteria. J. Gen. Microbiol. 72565-574. Zhou, J. C., and Y. Z. Cao. 1983. Nutrient requirements of the fast-growing rhizobia. J. Huazhong Agric. College 3:44-57. (In Chinese .) Zhou, J. C., Z. M. Zhang, C. J. Huang, F. D. Li, and H. K. Chen. 1987. Studies on Rhizobium plasmids. 11. Tests of plasmid elimination of the rhizobia from Astragalus sinicus. J. Huazhong Agric. Univ. 6:156-164. (In Chinese.)
