Diversity of Rhizobium Bacteria Isolated from the Root Nodules of Leguminous Trees

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INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Jan. 1991, p. 10&113 0020-7713/91/010104-10$02 .oo/o Copyright 0 1991, International Union of Microbiological Societies

Vol. 41, No. 1

Diversity of Rhizobium Bacteria Isolated from the Root Nodules of Leguminous Trees

XIAOPING ZHANG'T R. HARPER,' M. KARSIST0,1*2 AND K. LINDSTROM'" Department of Microbiology, University of Helsinki, SF-00710 Helsinki, Finland,' and

Department of Botany, University of Khartoum, Khartoum, Sudan2

Sixty rhizobial strains isolated from the root nodules of Acacia senegal and Prosopis chilensis in the Sudan were compared with 37 rhizobia isolated from woody legumes in other regions and with 25 representatives of recognized Rhizobium species by performing a numerical analysis of 115 phenotypic characteristics. Nineteen clusters were formed below the boundary level of 0.725 average distance, which was the level that separated the reference Rhizobium and Bradyrhizobium species. Our results indicated that tree rhizobia are very diverse with respect to their cross-nodulation patterns, as well as their physiological and biochemical properties, since 12 of the clusters formed consisted of tree rhizobia alone. Two distinctive features of tree rhizobia isolated in the Sudan were their high maximum growth temperature and their high salt tolerance.

Leguminous trees are abundant in savannah and arid regions of Australia, Africa, South America, North America, and Southeast Asia, where they grow in barren soils and dry sites that are unsuited for most crops. They provide fodder, firewood, and gums and protect the soil from erosion (26). Most leguminous trees form nodules and fix nitrogen in symbiosis with root nodule bacteria (3, 4, 11, 15, 25, 28, 29).

Early reports indicated that the microorganisms which were able to infect leguminous trees were slow-growing rhizobia and referred to them as Bradyrhizobium cowpea type (1-3, 12). However, later studies revealed that leguminous trees are infected as much by fast-growing rhizobia as by slow-growing rhizobia. Some tree rhizobial strains are host specific, whereas others have a wide host range (8, 10, 16, 31). Many strains even effectively nodulate herbaceous legumes (16, 30). Thus, it is improper to classify all tree strains as cowpea miscellany Bradyrhizobium species. On the basis of a small number of strains, it was suggested that the fast-growing tree strains are related to Rhizobium meliloti and Rhizobium leguminosarum (8,24,31). However, it is obvious that further studies will be necessary to determine the exact taxonomic position of the tropical tree rhizobia within the family Rhizobiaceae.

In this work we used numerical taxonomy to study the diversity of rhizobial strains isolated from the root nodules of Acacia senegal and Prosopis chilensis in the Sudan. We also compared the Sudanese strains with rhizobia which nodulate other woody legumes in different regions and with reference strains belonging to recognized Rhizobium species and to the genus Bradyrhizobium. By choosing appropriate tests, we especially looked for ecologically important physiological and biochemical properties of the isolates from the Sudan in order to select good inoculant strains for applica- tion in Sudanese silviculture.

MATERIALS AND METHODS

Bacterial strains. A total of 122 strains were used in this study; 60 strains were isolated from the root nodules of Acacia senegal and Prosopis chilensis in the Sudan, 37

* Corresponding author. t Present address: Agro-Chemistry Department, Sichuan Univer-

sity, Yaan, Sichuan, People's Republic of China.

strains were obtained from various leguminous trees growing in the tropical regions of Kenya, Thailand, Malaysia, Singa- pore, and the United States, and the other 25 strains were representatives of Rhizobium species and Bradyrhizobium cowpea type. The Sudanese isolation sites are described in Table 1, and all of the strains which we used are listed in Table 2. The purity of the cultures was verified by repeated streaking of single-colony isolates onto yeast extract-manni- to1 (YEM) agar (32). All strains were maintained in YEM broth containing 20% (vol/vol) glycerol at -80°C.

Phenotypic features. The biochemical and physiological tests were done in triplicate. The test cultures were incu- bated at 28"C, except the cultures used to determine maximum growth temperature (TmaX). Cultures were grown to log phase in YEM agar or broth before inoculation. The results were scored after 4 days for fast growers and after 10 days for slow-growers, unless indicated otherwise. According to Bergey 's Manual of Systematic Bacteriology (20), fast- growing root nodule bacteria (genus Rhizobium) form visible colonies (diameter, 2 to 4 mm) on YEM agar within 3 to 5 days at 25 to 3WC, whereas the slow-growing bacteria (genus Bradyrhizobium) form small (diameter, less than 1 mm) colonies within 5 to 7 days under the same conditions.

Growth on YEM agar. Strains were streaked onto YEM agar plates, and the length of time required for the first visible colony to appear was recorded.

Growth at pH 5.5 and 8.5. The ability to grow in acid and alkaline media was tested by inoculating strains onto YEM agar plates in which the pH was adjusted to 5.5 and 8.5, respectively.

Hydrolysis of urea. The appearance of a red color on Y EM agar plates amended with 2% (wt/vol) urea and 0.012% phenol red indicated that urea was hydrolyzed (24).

TABLE 1. Geographical and meteorological data about the rhizobial isolation sites in the Sudan

Location Latitude Longitude Mean annual Mean annual (N) 03 temp ("C) rainfall (mm)

Khartoum 15" 15' 32" 35' 28.5 160 Tendelti 13" 0' 31" 50' 25.5 350 El Obeid 13" 10' 30" 13' 25.7 360 Kosti 13" 13' 32" 40' 25.4 400 El Fau 14" 5' 34" 15' 26.0 450

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VOL. 41, 1991 DIVERSITY OF RHIZOBIA FROM TREES 105

TABLE 2. Bacterial strains, clusters, and cross-nodulation results ~~~

Cross-nodulation ofb:

Strain Host plant

Tree rhizobia TTR 1 HAMBI 1486 (= TTR 16) HAMBI 1497 (= 'ITR 28)

R. leguminosanrrn HAMBI 458 HAMBI 1138 (= CC 275e) TAL 1819 MPI 8001

R. galegae HAMBI 1141 (= gall) HAMBI 1144 (= ga17) HAMBI 1174 (= 1261R) HAMBI 540 (= ATCC 43677)

Tree rhizobium strain MPI 3054 R. leguminosarum

HAMBI 1125 (= CB 596) MPI 6001 HAMBI 499 (= 175F1) HAMBI 1116 (= NZP 5459) TAL 182

Tree rhizobia HAMBI 1487 (= E04) HAMBI 1178 (= MPI 3034) HAMBI 1177 (= MPI 3033) MPI 3036 MPI 3037

HAMBI 1129 (= NZP 2213) HAMBI 1338 MPI 4001

HAMBI 1337 (= USDA 191)

R. loti

R. fredii

Prosopis chilensis Prosopis chilensis Acacia senegal

Te, Sudan KO, Sudan EF, Sudan

1 O E O O O O I O 1 1 0 0 0 0 0 0 0 1 O E O O O O E O

Trifolium pratense Trifolium repens Trifolium sp. Trifolium sp.

Finland New Zealand NiffAL, Hawaii United Kingdom

2 O O O O E O O O 2 O O O O E O O O 2 O O O O E O O O 2 O O O O E O O O

Galega oficinalis Galega oficinalis Galega orientalis Galega orientalis Sesbania punctata

New Zealand New Zealand Finland Finland Malaysia

3 0 0 0 0 0 0 0 1 3 0 0 0 0 0 0 0 1 3 O O O O O O O E 3 O O O O O O O E 3 0 0 0 0 0 0 0 0

Pisum sp. Pisum sativum VZ5.z faba Phaseolus vulgaris Phaseolus vulgaris

United Kingdom The Netherlands United States New Zealand NiffAL, Hawaii

4 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0

Acacia senegal Leucaena leucocephala Leucaena leucocephala Leucaena leucocephala Leucaena leucocephala

EO, Sudan New Guinea New Guinea United States Hawaii

4 O E E O O O O O 4 E E E O O O O O 4 O E E O O O O O 4 E O E O O O O O 4 E O I O O O O O

Lotus tenuis Lotus tenuis Lotus pedunculatus

New Zealand New Zealand New Zealand

5 O O O O O O E O 5 O O O O O O E O 5 O O O O O O E O

Glycine soja People's Republic of

People's Republic of China

China

6 0 0 0 1 0 0 0 0

HAMBI 1339 (= USDA 201) Glycine soja 6 0 0 0 1 0 0 0 0

Tree rhizobia HAMBI 1507 (= TAL 1525) Prosopis chilensis MIRCEN, Bangkok,

MIRCEN, Bangkok,

NiffAL, Hawaii Niff AL, Hawaii EO, Sudan

Thailand

Thailand

7 E O E O O O O O

TAL s82 Leucaena leucocephala 7 E E E O O O O O

TAL 82 TAL 1145

through 7: HAMBI 1506 (= TTR 40)

Tree rhizobia HAMBI 1476 (= TTR 2) TTR 29 HAMBI 1502 (= TTR 34) HAMBI 1483 (= TTR 10) HAMBI 1485 (= TTR 12) HAMBI 1486 (= TTR 13) HAMBI 1505 (= TTR 37) HAMBI 1484 (= TTR 11) HAMBI 1491 (= TTR 17) HAMBI 1488 (= TTR 14)

Tree strain related to clusters 1

Leucaena leucocephala Leucaena leucocephala Acacia senegal

7 E I E O O O O O 7 E O E O O O O O

1 0 0 0 0 0 0 0

Prosopis chilensis Acacia senegal Acacia senegal Prosopis chilensis Prosopis chilensis Prosopis chilensis Prosopis chilensis Prosopis chilensis Prosopis chilensis Prosopis chilensis

Te, Sudan Te, Sudan Te, Sudan KO, Sudan KO, Sudan KO, Sudan KO, Sudan KO, Sudan KO, Sudan KO, Sudan

8 1 0 0 0 0 0 0 0 8 O E O O O O O O 8 O E O O O O O O 8 E E O O O O O O 8 E E O O O O O O 8 E E O O O O O O 8 E E O O O O O O 8 E I O O O O O O 8 E I O O O O O O 8 E O O O O O O O

Continued on following page


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106 ZHANG ET AL.

TABLE 2-Continued

INT. J. SYST. BACTERIOL.

Cross-nodulation ofb:

Strain Host plant Cluster Geographical origin or source"

HAMBI 1492 (= TTR 18) HAMBI 1489 (= TTR 15) HAMBI 1482 (= TTR 9) HAMBI 1481 (= TTR 8) Wl(5)a

Tree strain related to cluster 8: HAMBI 1498 (= TTR 27)

Tree rhizobia HAMBI 1478 (= TTR 4) HAMBI 1479 (= TTR 5 ) HAMBI 1501 (= TTR 31) HAMBI 1480 (= TI'R 6) HAMBI 1499 (= TTR 30) HAMBI 1394 HAMBI 1393

and 9: HAMBI 1504 (= TTR 36)

through 9: HAMBI 1493 (= TTR 19)

13229 13239 13235 13459 13241 13232 13237 13460 THA 205 TAL 1000 THA 305 TAL 420

D8(3)e

GB2 GB3 GB4 GB5 GB6 TTR 3 TSOl TS04 E02 E03 E05 TTR 22 TTR 38 'ITR 42 TTR 39 MPI 3031 MPI 3004 MPI 3008 E07 E06



Bradyrhizobium sp.

Tree strain related to cluster 10:

Tree rhizobia

~

Prosopis chilensis Acacia senegal Acacia Senegal Acacia Senegal Prosopis glandulosa Prosopis chilensis

~~

KO, Sudan KO, Sudan EF, Sudan EF, Sudan United States KO, Sudan

~

8 8 8 8 8

I E O O O O O O E E O O O O O O E E O O O O O O O E O O O O O O 0 0 0 0 0 0 0 0 E E O O O O O O

Acacia senegal Acacia senegal Acacia senegal Acacia senegal Acacia senegal Prosopis chilensis Prosopis chilensis Prosopis chilensis

Prosopis chilensis

Acacia mangium Acacia mangium Acacia rnangium Acacia mangium Acacia mangium Acacia mangium Acacia mangium Acacia mangium Arachis hypogaea Arachis hypogaea Egna radiata Kgna radiata Prosopis gla ndulosa

Acacia senegal Acacia Senegal Acacia senegal Acacia senegal Acacia senegal Prosopis chilensis Acacia senegal Acacia senegal Acacia senegal Acacia Senegal Acacia senegal Prosopis chilensis Prosopis chilensis Prosopis chilensis Acacia senegal Leucaena leucocephala Brownea ariza Centrosema piumieri Acacia senegal Acacia senegal

EO, Sudan EO, Sudan KO, Sudan KO, Sudan EF, Sudan Kenya Kenya Kh, Sudan

Kh, Sudan

Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand NiffAL, Hawaii NifTAL, Hawaii NifTAL, Hawaii NiffAL, Hawaii United States

Kh, Sudan Kh, Sudan Kh, Sudan Kh, Sudan Kh, Sudan Te, Sudan Te, Sudan Te, Sudan EO, Sudan EO, Sudan EO, Sudan KO, Sudan KO, Sudan KO, Sudan EF, Sudan Malaysia Singapore Singapore EO, Sudan EO, Sudan

10 10 10 10 10 10 10 10 10 10 10 10

11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 12 12

E E O O O O O O E O O O O O O O E E O O O O O O E E O O O O O O E E O O O O O O E E O O O O O O E E O O O O O O E E O O O O E O

E I O O O O O O

O O O E O O O O O O O E O O O O O I O E O O O O O I O E O O O O O O O E O O O O O O O E O O O O 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 O O O E O O O O O O O E O O O O O O O E O O O O O O O E O O O O 0 0 0 1 0 0 0 0

O E O O O O O O E O O O O O O O O E O O O O O O O E O O O O O O O E O O O O O O E E O O O O O O O E O O O O O O O E O O O O O O O E O O O O O O O E O O O O O O O E O O O O O O E E O O O O O O E E O O O O O O O E O O O O O O E E O O O O O O E O E O O O O O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 E E O O O O O O O E O O O O O O

~~



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TABLE 2-Continued

Strain Host plant Geographical origin or source" Cluster

Cross-nodulation of':

rr, 2 9 .Y E 8

~ ~~

E08 HAMBI 1395 TS03 TAL 1429

TTR 7

HAMBI 1495 (= TTR 21) HAMBI 1496 (= TTR 23) TTR 41 HAMBI 1392 HAMBI 1396 MPI 3038 MPI 3040 HAMBI 1193 (= MPI 3039)

HAMBI 470 HAMBI 1150 (= U45) HAMBI 1167 (= Balsac)

Tree rhizobia TS02 MPI 3001 MPI 3002 MPI 3048 P1( 1l)d TTR 32 MPI 3019

E01 GB1 TTR 33

Tree rhizobia HAMBI 1503 (= TTR 35) HAMBI 1494 (= TTR 20) HAMBI 1497 (= TTR 25) TTR 24 TI'R 26

MPI 3003

Tree strain relatd to cluster 13:

Tree rhizobia

R. meliloti

Tree strains related to cluster 18

Tree strain related to cluster 19:

Acacia senegal Prosopis chilensis Acacia senegal Acacia senegal Acacia senegal

Acacia senegal Acacia senegal Acacia senegal Prosopis chilensis Prosopis chilensis Leucaena leucocephala Leucaena leucocephala Leucaena leucocephala

Medicago sativa Medicago sativa Medicago sativa

Acacia senegal Adenanthera pavonia Albizia falcata Parkia javanica Prosopis glandulosa Acacia seyal Erythrina glauca

Acacia senegal Acacia senegal Acacia senegal

Acacia senegal Acacia senegal Acacia senegal Acacia senegal Acacia senegal Andira inemis

EO, Sudan Kenya Te, Sudan Hawaii KO, Sudan

Kh, Sudan EF, Sudan EF, Sudan Kenya Kenya Hawaii Hawaii Hawaii

Finland Uruguay Canada

Te, Sudan Singapore Singapore Singapore United States EF, Sudan Singapore

EO, Sudan Kh, Sudan EF, Sudan

Kh, Sudan KO, Sudan EF, Sudan EF, Sudan EF, Sudan Singapore

12 O E O O O O O O 12 E O E O O O O O 13 O E E O O O E O 13 O E E O O O E O

E E O O O O O O

14 I E E O O E O O 14 E E E O O I O O 14 E E E O O I O O 14 E E E O O O O O 15 E E E O O O O O 15 E E E O O E O O 15 E E E O O E O O 15 E E E O O O O O

16 O O O O O E O O 16 O O O O O E O O 16 O O O O O E O O

17 0 0 0 0 0 0 0 0 17 0 0 0 0 0 0 0 0 17 0 0 0 0 0 0 0 0 17 0 0 0 0 0 0 0 0 17 0 0 0 0 0 0 0 0 18 0 1 0 0 0 0 0 0 18 0 0 0 0 0 0 0 0

1 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 E E O O O O E O

19 E E E O O O E O 19 E E E O O O O O 19 E E E O O O O O 19 E E E O O O O O 19 I E E O O O O O

1 0 0 0 0 0 0 0

~ ~~~

" Khartoum (Kh), Tendelti (Te), El Obeid (EO), Kosti (KO), and E l Fau (EF) are the five locations in the Sudan from which strains were isolated (Table 1). NifTAL, Nitrogen Fixation for Tropical Agricultural Legumes; MIRCEN, Microbiological Resource Center.

E, Effective nodules formed; I, ineffective nodules formed; 0, no nodules formed.

Precipitation of calcium glycerophosphate, The formation of a white zone in Hofer medium (17) was recorded as a positive reaction for precipitation of calcium glycerophosphate.

Growth in the presence of 8% KNO,. Strains were tested for their ability to grow in the presence of 8% KNO, in YEM agar (9).

Reduction of nitrate, Reduction of nitrate to nitrite was tested in liquid medium as described previously (24).

Production of melanin. Melanin production by the strains was detected by using a modification of the procedure described by Cub0 and coworkers (7). In the modified

method, 100 pg of L-tyrosine per ml instead of 600 pg/ml was added to the medium.

NaCl tolerance. The NaCl tolerance of the strains was tested by growing them in tryptone-yeast extract (TY) agar (5) containing 0, 1, 2, and 3% NaCl. A multipoint inoculator that fit a 96-well microdilution tray was used to spot inocu- late strains onto large plates (diameter, 14 cm) (6).

Intrinsic heavy metal resistance. The resistance of strains to heavy metals was determined on solid TY medium. The stock solutions of metals were filter sterilized and added to sterile TY agar as follows: CuCl, . 2H,O, 100 pg/ml; AlCl, . 6H,O, 500 pg/ml; HgCl,, 5 pglml; CdC1, . 2H,O, 20


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108 ZHANG ET AL. INT. J. SYST. BACTERIOL.

pg/ml; ZnCl,, 100 pg/ml; and Pb(CH,COO),, 500 pg/ml. The inoculation method used was the same as that described above for the NaCl tolerance test.

Intrinsic antibiotic resistance. The intrinsic antibiotic resistance test was performed by using TY agar to which one of the following antibiotics (Sigma) was added: streptomycin (3 pg/ml), lincomycin hydrochloride (100 pg/ml), spectinomycin (5 pg/ml), rifamycin (10 pg/ml), neomycin sulfate (10 pglml), trimethoprim (200 pg/ml), novobiocin (5 pg/ml), kanamycin (10 kg/ml), chloramphenicol (15 pg/ml), and erythromycin (30 pg/ml). The antibiotic solutions were filter sterilized and added to the medium after the medium was autoclaved and cooled to approximately 55°C. The inoculation method used was the same as that described above for the NaCl tolerance test.

Utilization of amino acids as sole nitrogen sources. The ability to utilize each of the following 19 amino acids as a sole nitrogen source was tested on Def 8 agar medium (24) from which sodium glutamate was omitted: DL-valine, L-glutamic acid, L-( -)-tyrosine, DL-leucine, L-proline, DL-threonine, L-( +)-isoleucine, DL-phenylalanine, DL-tryptophan, DL-lysine, glycine, DL-serine, L-histidine, L-arginine, L-me- thionine, L-alanine, L-asparaginic acid, and L-( -)-asparagine. Def 8 medium containing sodium glutamate was used as a positive control, and Def 8 medium without any nitrogen source was used as a negative control. The stock solutions were filter sterilized, and the amino acids were added to Def 8 medium to final concentrations of 10 mM. The inoculation method used was the same as that described above for the NaCl tolerance test.

Utilization of carbon sources. Utilization of various carbon sources was tested on Def 9 agar, which is similar to Def 8 medium but contains 1.0 g of NH,C1 per liter instead of sodium-glutamate as a nitrogen source and no carbon source (24). Stock solutions were filter sterilized, and the carbon sources were added to Def 9 medium to final concentrations of 0.2%; 0.0025% bromothymol blue was used as a pH indicator. The pH was adjusted to 7.0 to 7.2 by using sterile 1 N NaOH or 1 N HC1 after the carbon source was added. The following 52 carbon sources were tested: caprate, starch, itaconate, glycerol, nicotinate, L-arabinose, aconi- tate, D-galactose, dulcitol, sodium formate, sodium acetate, amygdalin, arbutin, sorbitol, sodium citrate, meso-inositol, sodium butyrate, inulin, dextrin, raffinose, D-melezitose, L-rhamnose, maltose, lactose, D-fructose, D-mannose, 0-methyl-D-mannoside, a-ketoglutarate, sodium maltose, trehalose, D-( +)-melibiose, sodium lactate, D-ribose, xylose, esculin, amyl-alcohol, polyethylene glycol, anthrone, salicin, cholesterol, ergosterine, vanillin, riboflavin, xylene, (-)-camphor, propanol, menthol, glucuronic acid, adipic acid, choline chloride, 1,2-propylene glycol, and 1 ,2-butylene glycol. Def 9 medium containing mannitol was used as a positive control, while Def 9 medium without any carbon source was used as a negative control. The strains were inoculated by using the method described above for the NaCl tolerance test.

T,,,. T,,, values were determined by using a model MAX-7B temperature gradient incubator (a prototype con- structed by s. I. Niemela) (27). The TY agar in the gradient was inoculated by filtering the inoculum onto a nitrocellulose membrane through a right-angle funnel and transferring the membrane onto the medium. Incubation was for 1 to 2 h at room temperature, followed by 2 to 10 days in the temperature gradient. The temperature range in the gradient was 31 to 44°C.

Cross-nodulation. Each of the strains tested was grown on

YEM agar plates and inoculated onto three leguminous tree species (Acacia senegal, Prosopis chilensis, and Leucaena leucocephala K636) and five herbaceous legumes (Macrop- tilium atropurpureum, Lotus corniculatus, Trifolium pratense cv. Venla, Medicago sativa cv. Iroquois, and Galega orientalis). Trees were grown in plastic pots (diameter, 7 cm; height, 14 cm) containing a mixture of sand and vermiculite in a growth chamber (25) and were watered with a 1:4 dilution of Jensen nitrogen-free solution (32). The herbs were planted in Jensen agar tubes (diameter, 1.7 cm; height, 15 cm). Nodulation was checked after 8 weeks for the trees and after 4 weeks for the herbs, Each of the treatments was set up in triplicate. Results were recorded as positive (nodules were found) or negative (no nodules were found). The acetylene reduction method was used to evaluate the effectiveness of nodulation (22, 23).

Numerical analysis. Characters were coded 1 for positive and 0 for negative. The Tmax values were divided into groups by using the following temperature ranges: 31 to 37,38 to 40, 41 to 43 and 44°C. The final matrix contained 122 strains and 115 features. Cluster analysis was carried out by using the single-linkage , complete-linkage , and average-linkage methods, Ward minimum variance cluster analysis, Mcquitty similarity analysis, and centroid and median hierarchical cluster analyses (SAS program, VAX VMS, University of Helsinki). The results obtained with the average-linkage clustering method are presented in Fig. 1 because this method gave the best separation of the reference strains.

RESULTS

All but 10 rhizobial strains were included in 19 distinctive clusters formed at the boundary level of 0.725 average distance, which was the level that divided the reference strains of R . leguminosarum, R . meliloti, Rhizobium loti, Rhizobium fredii, Rhizobium galegae, and Bradyrhizobium cowpea type into separate clusters (Fig. 1). The composition of each cluster is shown in Table 2, which also shows the cross-nodulation patterns of the strains. The physiological and biochemical properties of the clusters are shown in Table 3.

Cluster 1 consisted of three Sudanese strains, two of which also nodulated Lotus corniculatus. The T,,, values of these strains were high, but otherwise the strains resembled recognized Rhizobium species.

Clusters 2 through 6 represented R . leguminosarum biovar trifolii, R. galegae, R . leguminosarum biovar viciae and R . leguminosarum biovar phaseoli, R. loti, and R . fredii, respectively. One tree strain was included in the R. galegae cluster (cluster 3). Five tree rhizobia, one of which was Sudanese, were clustered together with R. leguminosarum in cluster 4. A characteristic feature of the latter group of tree rhizobia was that they nodulated Leucaena leucocephala in addition to Acacia sp. or Prosopis sp. or both. The tree rhizobia in cluster 7 shared this property with the tree strains in cluster 4.

Clusters 1 through 7 plus strain TTR 40 formed a larger group with properties similar to those generally found for the Rhizobium species which belonged to it, such as low levels of resistance to NaCl and most heavy metals and low T,,, values. This larger group contained only five Sudanese strains.

Clusters 8 and 9 together with strains TTR 27, TTR 36, and TTR 19 formed a larger group which was almost entirely composed of Sudanese isolates. Most of the strains in these clusters nodulated only Acacia sp. or Prosopis sp. or both;


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-===I -m33GB 18 n a ( 2 ) Eo

1 1 TrR(18)

- W8)e

10 Bradyhzobium sp. (12)

8 'ITR(15)

4 Rhizobium leguminosarum + 'ITR (5+5)

3

2 Rhizctiumlegmhmrum(4)

1 'ITR(3)

Rhizobium galegae + 'ITR (4+1)

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0

FIG. 1. Dendrogram showing the phenotypic relationships among the strains of Rhizobium and Bradyrhizobium species investigated based on average-linkage cluster analysis of 115 characteristics. The average distances between the clusters are shown on the x axis; the number of strains included in a cluster is shown in parentheses.

the single exception was the isolate from Prosopis glandu- tosa obtained from the United States, which did not nodulate any of the test plants. The T,,, values of all of the isolates were above 38"C, but their levels of salt tolerance were only moderate. A majority of the strains were melanin producers. The range of carbon compounds utilized was fairly narrow.

Cluster 10 included four strains of the cowpea type and eight tree rhizobia isolated from the root nodules of Acacia mangium in Thailand. In our experiments these organisms nodulated Macroptilium atropurpureum, but not the three

tree species tested. They showed visible growth after 7 days of incubation at 28°C. Their levels of intrinsic antibiotic resistance were generally high, and the ranges of carbon compounds utilized were narrow. Thus, they displayed features typical of slow growers (Bradyrhizobium sp.). Their T,,, values were below 38"C, and they showed little resistance to NaC1. Cluster 10 plus strain DS(3)e from Prosopis glandulosa (obtained from the United States) formed a group which was not closely related to the fast growers.

Clusters 11 and 12 comprised 22 strains, all but three of which came from the Sudan. These organisms nodulated both Acacia senegal and Prosopis chilensis. Two strains also nodulated Leucaena leucocephala. All of the strains grew at pH 8.5, 19 strains were resistant to 3% NaC1, and 16 strains grew at 41 to 43°C. The levels of heavy metal resistance and intrinsic antibiotic resistance of these strains were higher than the levels for clusters 1 through 9, and the ranges of amino acids and carbon compounds used for growth were broader. Ten strains in cluster 11 produced melanin, but none of the strains in cluster 12 had this feature.

The two strains in cluster 13 nodulated Acacia senegal and Leucaena leucocephala but not Prosopis chilensis. More interestingly, they formed effective nodules on the roots of Lotus corniculatus. Their T,,, values were in the 38 to 40°C range, and they tolerated 2% NaC1. They could also utilize a wide range of carbon compounds.

Clusters 14 and 15 included eight tree rhizobial strains which were from the Sudan, Kenya, and Hawaii. These organisms were very similar to the strains in cluster 16, which were classified as members of R . meliloti. The general features of these clusters were as follows: moderate to high T,,, values and high levels of salt and metal tolerance. Strain TTR 23 even grew at 44°C. The tree rhizobia effectively nodulated Acacia senegal, Prosupis chilensis, and Leucaena leucocephala. Five tree rhizobia also nodulated Medicago sativa.

The five strains in cluster 17 were isolated from different tree species in the Sudan, Singapore, and the United States. They did not nodulate any of the tree species tested, but metabolically they were related to strains in clusters 11 through 16.

The two strains in cluster 18 together with strains E01, GB1, and TTR 33 shared some distinctive features, which placed them in loose association with clusters 11 through 17. These strains came from the Sudan and from Singapore and had diverse nodulation patterns. They precipitated calcium glycerophosphate and reduced nitrate. Their levels of heavy metal resistance and intrinsic antibiotic resistance were remarkably high, they tolerated 3% NaC1, their T,,, values were 41 to 43"C, and they could utilize a wide range of nitrogen and carbon compounds.

All five strains in cluster 19 isolated in the Sudan formed effective nodules on Acacia senegal, Prosopis chilensis, and Leucaena leucocephala, and one strain also nodulated Lotus corniculatus. In our tests these organisms hydrolyzed urea and precipitated calcium glycerophosphate. Four strains reduced nitrate, and three strains grew in the presence of 8% KNO,. Their levels of heavy metal resistance and salt tolerance were remarkably high, but the levels of intrinsic antibiotic resistance were generally lower than those for members of cluster 18. Their T,,, value was 38 to 40°C. The cluster 19 strains shared the ability to utilize a wide range of nitrogen sources with cluster 18 strains, but the spectrum of carbon sources used was different from the spectra of carbon sources used by the members of all other clusters. Together


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TABLE 3. Results of biochemical and metabolic tests for the clusters formed by using the average-linkage clustering method

Characteristic

U m h

II t Y

rl

h d II t c.l v

h 10 I1 P m v

h

3 II E d v

h d II 5 l-

h

3 II E

00 v

h l-

II 3

h

00 rl

II P + rl

v

n c.l

II t

m rl

V

h d II t V

f

h d II E: V

3

h m II t v

2

s II t v

2

h 10

II E

m rl

v

First colony formed after: 1-4 days + b + + + + + + + + - + + + + + + + + + 7-10 days -

Growth at pH 8.5 1 - 4 2 - + - 3 2 1 + + + 3 + + 4 + +

- - - - - - - - + - - - - - - - - - 2 1 - - 1 - - - - - 1 1 3 Growth at pH 5.5

I - - - - - - 1 + Hydrolysis of urea 3 + + Precipitation of calcium

2 1 - - - - - - 1 3 Growth in the presence of

Reduction of nitrate - + 1 8 - - 2 2 1 + - - 1 - - + 4 + 4 Melanin production + - - 1 - 1 - 1 1 6 - 1 0 - - 2 2 + 1 1 - NaCl tolerance

1% + - 1 5 - 1 - + 3 1 + + + + + + + + + 2% 1 - - I - - - 1 2 - + 3 1 + + + - + +

16 1 - 1 + + - + + 3%

- - - - - -

- - - - - - - - - - - - - - - - I - - - 1 3 - - - -

- glycerophosphate

8% KNO, - 1 - - - - - -

- - - - - - - - - - Intrinsic heavy metal resis-

tance Pb 1 2 4 4 1 + 1 1 4 + - 17 + + + + + + 1 + Zn - 3 + 1 2 - - - - 7 1 1 1 1 - 3 1 4 + + Cd - 2 4 1 2 - - - 1 - 17 - 1 1 + 2 3 + +

1 6 - - 1 1 - 1 + + Hg c u 2 2 2 3 - 1 1 1 4 2 - 17 + 1 + + + + + + Al - - I - - - 2 - - 4 1 0 3 1 - 3 - + + 4

Streptomycin 1 2 + 2 2 - 2 1 2 1 1 + - - 1 3 2 + + + Lincomycin + 2 + 4 - + - + + 11 + + 1 + + + + + 1 Spectinomycin - - 3 1 2 - - - - 6 + + - + + + 4 + + Rifampin - - 1 5 - - - - 1 8 3 2 - - 2 2 4 + -

1 + 1 Neomycin Trimethoprim + + + 6 - 1 2 5 4 7 1 6 3 - + 3 + + 1 - Novobiocin - - 1 2 - - - 7 3 1 + + - + 3 2 4 + + Kanamy cin 1 - + 4 + - 2 - - 2 + - 1 1 1 + + + + Chloramphenicol 1 1 - 2 - 1 - - 1 7 1 6 1 - + + + 4 + - Erythromycin 1 - 1 - + 1 2 1 2 - + + + + + + 4 + -

- 1 - - - - - - - -

Intrinsic antibiotic resistance

- - - 1 - - - - - 8 3 - - - - -

Utilization of the following amino acids as sole nitrogen sources:

- + + DL-Valine 2 - - - - - - 16 + - 1 + + + + + L-Glutamic acid

- 1 + L-( -) -Tyrosine - - - - - - - - - 3 - - - - - 2 - 2 DL-Leucine -

L-Proline + + + + + + + + + + + + + + + + + + + - + 4 DL-Threonine

10 3 - 1 - - 4 + + L-( + )-Isoleucine - + + DL-Phenylalanine - 1 + DL-Tryptophan - 1 + DL-Lysine - + + Glycine - + + DL-Serine

- - - _ - - _ _ - _ _ _ _ _ _ _ - - - - - - _ - _ _ _ - _ _ _ _ _ _ _

- - - - - - - - - - 1 - _ _ _ _

- - - - - - - - - - 1 - - - _ - - - - - - - - - - -

- - - - - - - _ - - - - - - _ _ - - - _ - - _ _ - _ _ _ _ _ _ _ - - - - - - - - - - - - - - _ _ - - - - - - _ _ - _ 1 _ _ _ _ _

L- Arginine + + + + + + + + + + + + + + + + + + + Sodium glutamate + + + + + 1 + + + - + + + + + + + + + L-Histidine + + + + + + + + + + + + + + + + + + + I.-( + )-Asparagine L-( -)-Asparagine L-Alanine L-Me thionine

- - - - - - - - - - - - - _ _ _ _ _ _ - - - - - - - - - - - - - _ _ _ _ _ _ - - - - - - - - - - - - _ _ _ _ _ _ _ - - - - - - - - - - - - - _ _ _ _ _ _

Utilization of the following carbon sources:

2 1 - - 6 3 1 - 1 - 2 1 4 Caprate Starch 2 2 - - - 1 2 2 6 - 1 7 3 - 1 - - - - - Itaconate + 2 + 7 + 1 3 2 3 9 + + + + 3 + + + +

- - - - - -



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VOL. 41, 1991

TABLE 3-Continued

DIVERSITY OF RHIZOBIA FROM TREES 111

Characteristic

El R II t w

4

h

m II E m v

R II r w

m

s II E \o V

5 I1 E

00 V

3 I1 t v

2

h m II e W

2

s II s 00 r(

h

m II s m 4

Glycerol Nicotinate L- Arabinose Aconi ta te D-Galactose Dulcitol Sodium formate Sodium acetate Amygdalin Arbutin Sorbitol Sodium citrate rneso-Inositol Sodium butyrate Inulin Dextrin Raffinose D-Melezitose L-Rhamnose Maltose Lactose D-Fructose D-Mannose 0-Methyl-D-mannoside a-Ketoglutarate Sodium maltose Trehalose D-( + )-Melibiose D-Ribose Xylose Esculin Amyl-alcohol Polyethylene glycol Anthrone Salicin Cholesterol Ergosterine Vanillin Riboflavin Xylene (-)-Camphor Propanol Menthol Glucuronic acid Adipic acid Choline chloride 1,2-Propylene glycol 1,2-Butylene glycol

31-37°C 3840°C 4143°C 44°C

T,,

~~~ ~

- 1 + 5 - - 1 - - 1 1 7 3 - 1 2 + 4 + 1 4 + 4 - - - - - - - - - - 5 3 - - - -

+ + + + + + 3 2 + + + 4 + + + + + + - 2 + 4 2 - 1 + 7 - + 13 2 1 1 - 2 - + - + + + + + + + + + + + + + + + + + + l + + 3 6 - + 3 5 3 - + 4 + 1 + + 2 + - - - 1 - - - - - - - 11 3 + - 1 - + + +

5 3 1 - 1 - 4 + + + + + + + + + 5 4 + + 4 + 3 + + + + - + + + + + + + 2 + 9 + + + + 3 + + + 1 + + + + + + + 1 2 + + + + + + + + + + 2 + 3 9 + - + 1 4 + - + + + + + + + + + + + + + + + + + + + - + + + + + + + + 1

1 + + + + + + 2 - 4 9 + 10 + + + + + + 4 1 - + + + + + + 4 1 4 + - + + + + + + + + 1 + + + + 2 + + 1 6 - + + + 4 + + + 1 - + + 4 4 2 - 4 1 3 - 9 3 + 2 2 + - - 2 + + + + + + + 14 6 - + + + + + + + + - + + + + + + + 2 + - + 4 + + 3 + + + 1 + - + + + + + 2 + - + 4 + + + + + + 1 + + + + + + + 4 6 7 + + + + + + + + 1 + + + + + + + 5 + 9 + + + + 3 + + + - + + + + + + + + + + + + + + + + + + - + + + + + + + + + 8 + + + + + + + + + + + + + + + + + + 7 + + + + + + + + + + + + + + + + 6 6 1 + + + + 3 + + 1 1 + - - - - + - - - - 16 2 + - + - - - - + + + + + + + 8 + - + + + + + + + 1 1 + 3 + + - + + 4 4 7 1 7 + + + + + + + - 2 + + + 1 - + - 1 - 16 - + + 2 - 4 + 1 _ _ _ _ - - - I - - 1 I - - - - 1 + 4 1 2 4 1 - - 1 1 3 - 17 + + 1 - - - - -

+ + 3 3 - - 1 - - - 1 7 + + - - + - 1 - + + 3 2 - - 4 - 2 - 17 1 1 3 2 + 1 - - + + 2 - - - 2 - 2 - 1 6 3 + - 2 - - - - + 2 4 - - - 1 1 2 - 17 + + 1 1 - - 1 - _ _ _ _ _ _ 2 - - - 2 1 - - - - 1 1 2 1 - 4 - - - - 1 1 - 1 7 3 + - - - - - -

+ + + 1 - + 1 3 2 5 + + + 1 1 - - 1 - 2 3 + - - - 1 1 1 - 17 3 + - - - 1 - - + - + 3 2 - 1 1 2 2 1 2 + - - - - - 1 - 2 2 1 - - - - - 3 - 17 + + 1 2 2 2 - - - - + 1 - - 4 - 1 1 1 6 + + - 1 + + 1 + - - 4 1 + 1 + 3 - + 15 + + - 3 + + + + - - 3 1 2 - 2 1 1 - 17 3 1 - + 2 + + + + + + + + + + 2 5 1 1 + + + 3 + + + + + 2 + + + + + + 2 2 1 1 + + + + + + + + +

_ _ - _ - - - - - -

- - - - - - - - - - - - - - - -

- 1 + 7 + + 3 - - + - - I - - - 1 - 3 1 3 - 3 - - 1 1 2 7 - 5 2 1 1 - + 4 - 2 2 - - - - - - 3 1 - 1 0 2 - 1 + - - + - _ - - - - - - - - - - - - I - - - - -

a n is the number of strains tested. ’ +, All strains were positive; -, all strains were negative. The numbers are the numbers of positive strains.


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with strain MPI 3003 cluster 19 formed a separate unit on the dendrogram.

DISCUSSION

Rhizobia which nodulate leguminous trees are fast or slow growing (10). The strains isolated from Acacia senegal and Prosopis chilensis in the Sudan were all fast growing. On the other hand, the strains isolated from the root nodules of Acacia mangium growing in Thailand in this study were slow growing (Table 3).

Dommergues et al. (8) summarized recent findings and proposed that nitrogen-fixing trees could be divided into the following three broad groups according to their nodulation patterns with fast- and slow-growing tree rhizobia: group 1, which forms nodules with fast-growing strains; group 2, which forms nodules with both fast- and slow-growing strains; and group 3, which forms nodules with slow-growing strains. Accordingly, we propose that Acacia senegal, Prosopis chilensis, and Leucaena leucocephala belong to group 1, since they were nodulated only by fast-growing tree rhizobia (Table l) , and that Acacia mangium belongs to group 3. This proposal was further confirmed by our repeated failure to isolate slow growers from the root nodules of Acacia senegal and Prosopis chilensis and fast growers from Acacia mangium. Of the three strains obtained from the United States and isolated from the same host (Prosopis glandulosa), one [strain D8(3)e] was slow growing and the other two [strains Wl(5)a and Pl(ll)d] were fast growing (19). Thus, Prosopis glandulosa belongs to group 2.

In our study, isolates from Acacia senegal, Prosopis chilensis, and Leucaena leucocephala cross-infected each other's host plants (Table 1). Consequently, these tree species are in the same cross-nodulation group. However, strains isolated from Sesbania punctata, Brownea ariza, Centrosema plumieri, Prosopis glandulosa, Adenanthera pavonia, Albizia falcata, Parkia javanica, Erythrina glauca, and Acacia mangium did not nodulate the three tree species tested. Because of a lack of seeds, the hosts of these strains were not used in the cross-nodulation study. Some fast- growing tree rhizobia infected Lotus corniculatus (clusters 1, 13, and 19) and Medicago sativa (clusters 14 and 15). All slow growers and the cowpea type strains nodulated Mac- roptilium atropurpureum. Our results agree with those of previous studies, confirming that the nodulation capabilities of the tree rhizobia are diverse. Some strains have a narrow host range, whereas others have a broad host range (10, 11, 13, 14, 16, 21, 31). The observation about nodulation of Medicago sativa by tree rhizobial strains is in agreement with similar findings of Trinick (30) and Herrera et al. (16).

The dendrogram in Fig. 1 shows that there is a high level of diversity among the fast-growing tree rhizobia. Of the 19 clusters formed, 12 were composed of tree rhizobia alone. These clusters showed no relatedness to each other or to the reference strains at the boundary level of 0.725 average distance. In addition, one tree strain was grouped together with R . galegae (cluster 3), and five tree strains were grouped together with R. leguminosarum (cluster 4). The taxonomic method which we used could not separate members of the genus Rhizobium from members of the genus Bradyrhizobium at the generic level. The reasons for this were probably the narrow selection of Bradyrhizobium strains used and the fact that the experiments were designed in such a way that an emphasis was put on the special features of the Sudanese rhizobia.

The diversity of the fast-growing tree rhizobia was not

merely a consequence of their different geographical origins or different host plants. The clusters formed by the strains isolated from Acacia senegal and Prosopis chilensis at five different locations in the Sudan were not correlated with the isolation site or host (Table 1). On the contrary, the Sudanese tree strains were quite evenly distributed among all clusters that harbored tree rhizobia. The clusters formed by the strains isolated from various trees in other regions were not correlated with isolation site or host either.

When the Sudanese tree rhizobia are compared with the tree rhizobia from other regions, it is possible to identify differences in cross-nodulation patterns, which presumably reflect the different isolation hosts. In addition, the tree rhizobia from the Sudan showed a greater ability to tolerate salt and heavy metals than the other strains, and their T,,, values were high.

The taxonomic position of leguminous tree rhizobia within the Rhizobiaceae has long been discussed. Although two recent reports have suggested that the fast-growing tree rhizobia resemble R . meliloti and R . leguminosarum (24,31), these bacteria are still often referred to as belonging to the cowpea miscellany, simply because they often also nodulate cowpeas (13, 14). Our results confirmed the differences between the fast growers and the slow growers belonging to the cowpea miscellany. Most of the fast growers were not related to any of the reference Rhizobium species, although a few strains showed some relationship to R . meliloti, R . leguminosarum, and R . galegae. In order to determine the exact taxonomic position of leguminous tree rhizobia, further work on bacterial genomic properties is especially needed. The only group that has been investigated so far is the Leucaena rhizobia. Jarvis (18) found that these rhizobia could be divided into three different DNA homology groups. In this study, Leucaena rhizobia were found in four different clusters. It seems that the fast-growing tree rhizobia stem from a reservoir of taxonomically diverse but ecologically well-adapted strains. The type of symbiotic plasmids which these strains carry determines their host range, which is analogous to the case of the three biovars of R. leguminosarum (33).

Graham and Parker (12) reported that most Rhizobium species can tolerate up to 2% NaCl in the growth medium. In our study, a large number of fast-growing tree rhizobia from the Sudan were found to grow normally even at NaCl concentrations of 3%. Similar observations were also made by Basak and Goyal (4) with isolates from Acacia species. Another unusual feature about the Sudanese tree rhizobium strains was their abnormally high T,,, values, which ranged from 38 to 44°C. According to the results obtained by Lindstrom and Lehtomaki (24), the T,,, of R . leguminosarum is 31 to 35"C, the T,,, of R. meliloti is 38 to 41"C, the TmaX of R . loti is 33 to 37"C, the T,,, of R . fredii is 34 to 36"C, and the T,,, of R . galegae is 33 to 37°C. On the basis of these data, the tree rhizobia from the Sudan are more closely related to R. meliloti than to other Rhizobium species. The higher T,,, values and levels of salt tolerance of these strains may play an ecological role under the hot dry conditions found in the Sudan.

ACKNOWLEDGMENTS

We express our gratitude to S. I. Niemela for his valuable advice on numerical analysis. Seeds of Leucaena leucocephala were kindly provided by The Nitrogen Fixing Tree Association, Waimanalo, Hawaii; seeds of Macroptilium atropurpureum were provided by Alan Gibson, CSIRO, Canberra, Australia; and the MPI strains were provided by W. J . Broughton, Max-Planck-Institut, Cologne,


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Federal Republic of Germany (present address, LBMPS, Cham- besy, Geneva, Switzerland). Other reference tree rhizobia were provided by M. B. Jenkins, University of California, Riverside; R. A. Virginia, San Diego State University, San Diego, Calif.; the NifTAL project and MIRCEN at Paia, Hawaii; and MIRCEN at the Department of Agriculture, Bangkhen, Bangkok, Thailand. The nodules of Acacia mangium were provided by Orapin Bhumib- hamorn, Kasetsart University, Bangkok, Thailand. The assistance of Juhani Polkunen, University of Helsinki, and Ahmed Ali Mahdi, Eshrak Mohammed Atahani, and Omaimi Abdallah Marhoum, University of Khartoum, is greatly acknowledged.

This work was supported by the Academy of Finland.

REFERENCES 1. Allen, 0. N., and E. K. Allen. 1936. Root nodule bacteria of

some tropical leguminous plants. I. Cross nodulation studies with Vigna sinensis. Soil Sci. 42:61-77.

2. Allen, 0. N., and E. K. Allen. 1939. Root nodule bacteria of some tropical leguminous plants. 11. Cross nodulation test with cowpea group. Soil Sci. 47:63-76.

3. Allen, 0. N., and E. K. Allen. 1981. The Leguminosae: a source book of characteristics, uses and nodulation. The University of Wisconsin Press, Madison.

4. Basak, M. K., and S. K. Goyal. 1980. Studies on tree legumes. 111. Characterization of the symbionts and direct and reciprocal cross inoculation studies with tree legumes and cultivated legumes. Plant Soil 56:39-51.

5. Beringer, J. E. 1974. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84:18&198.

6. Brockman, F. J., and D. F. Bezdicek. 1989. Diversity within serogroups of R . leguminosarum biovar viciae in the palouse region of eastern Washington as indicated by plasmid profiles, intrinsic antibiotic resistance, and topography. Appl. Environ. Microbiol. 55: 109-115.

7. Cubo, M. T., M. Boendia-Claveria, J. E. Beringer, and J. E. Ruiz-Sainz. 1988. Test for melanin production. Appl. Environ. Microbiol. 54: 1812-1813 -

8. Dommergues, Y. R., H. G. Diem, D. L. Gauthier, B. L. Dreyfus, and F. Cornet. 1984. Nitrogen-fixing trees in the tropics: poten- tialities and limitations, p. 7-8. In C. Veeger and W. E. Newton (ed.), Advances in nitrogen fixation research. Proceedings of the 5th International Symposium on Nitrogen Fixation. Martinus Nijhoff/Dr W. Junk Publishers, Wageningen, The Netherlands.

9. Dreyfus, B., J. L. Garcia, and M. Gillis. 1988. Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a stem- nodulating nitrogen-fixing bacterium isolated from Sesbunia rostrata. Int. J. Syst. Bacteriol. 38:8%98.

10. Dreyfus, B. L., and Y. R. Dommergues. 1981. Nodulation of Acacia species by fast- and slow-growing tropical strains of Rhizobium. Appl. Environ. Microbiol. 41:97-99.

11. Felker, P., and P. R. Clark. 1980. Nitrogen fixation (acetylene reduction) and cross inoculation in 12 Prosopis species. Plant Soil 57:177-186.

12. Graham, P. H., and C. A. Parker. 1964. Diagnostic features in the root nodule bacteria of legumes. Plant Soil 20:383-396.

13. Habish, H. A,, and S. M. Khairi. 1968. Nodulation of legumes in the Sudan: cross-nodulation groups and the associated Rhizo- bium strains. Exp. Agric. 4:227-234.

14. Habish, H. A., and S. M. Khairi. 1970. Nodulation of legumes and cross-nodulation of Acacia spp. Exp. Agric. 6:171-176.

15. Hansen, A. P., and J. S. Pate. 1987. Comparative growth and symbiotic performance of seedlings of Acacia spp. in defined

pot culture or as natural understory components of a eucalypt forest ecosystem in SW Australia. J. Exp. Bot. 38:13-25.

16. Herrera, A. M., E. J. Bedmar, and J. Olivares. 1985. Host specificity of Rhizobium strains isolated from nitrogen-fixing trees and nitrogenase activities of strain GRH2 in symbiosis with Prosopis chifensis. Plant Sci. 42:177-182.

17. Hofer, A. W. 1941. A characterization of Bacterium radiobac- ter. J. Bacteriol. 41:193-224.

18. Jarvis, B. D. W. 1983. Genetic diversity of Rhizobium strains which nodulate Leucaena leucocephala. Curr. Microbiol. 8: 15 3-1 5 8.

19. Jenkins, B. M., R. A. Virginia, and W. M. Jarrell. 1987. Rhizobial ecology of the woody legume mesquite (Prosopis glandulosa) in the Sonoran desert. Appl. Environ. Microbiol. 53:3640.

20. Jordan, D. C. 1984. Family 111. Rhizobiaceae Conn 1938, p. 234-244. I n N. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore.

21. Lange, R. T. 1961. Nodule bacteria associated with the indige- nous Leguminosae of South-West Australia. J. Gen. Microbiol.

22. Lindstrom, K. 1984. Analysis of factors affecting in situ nitrogenase activity of Galega orientalis, Trifolium pratense and Medicago sativa in temperate conditions. Plant Soil 79:32%341.

23. Lindstrom, K. 1984. Effect of various Rhizobium trifolii strains on nitrogenase activity profiles of red clover (Trifolium pratense). Plant Soil 80:79-89.

24. Lindstrom, K., and S. Lehtomaki. 1988. Metabolic properties, maximum growth temperature and phage sensitivity of Rhizo- bium sp. (Galega) compared with other fast growing rhizobia. FEMS Microbiol. Lett. 50:277-287.

25. Miettinen, P., 0. Luukkanen, S. Johansson, E. Eklund, and J. Mulatya. 1988. Rhizobium nodulation in Prosopis juliflora seedlings at different irrigation levels in eastern Kenya. Plant Soil

26. National Academy of Sciences. 1979. Tropical legumes: re- sources for the future. National Academy of Sciences, Wash- ington, D.C.

27. Niemela, S. I., J. Mentu, P. Vaatiinen, and K. Lahti. 1983. Maximum growth temperature as a diagnostic character in Enterobacteriaceae. INSERM Colloq. 114:619-627.

28. Roskoski, J. P., and T. Wood. 1984. Nodulation and nitrogen fixation by five species of leguminous trees grown in soil from undisturbed and disturbed tropical sites in Mexico. Adv. Agric. Biotechnol. 4:357.

29. Shoushtari, N. H., and I. L. Pepper. 1985. Mesquite rhizobia isolated from the Sonoran desert: physiology and effectiveness. Soil Biol. Biochem. 17:797-802.

30. Trinick, M. J. 1965. Medicago sativa nodulation with Leucaena leucocephala root-nodule bacteria. Aust. J. Sci. 27:263-264.

31. Trinick, M. J. 1980. Relationships amongst the fast-growing rhizobia of Lablab purpureus, Leucaena leucocephala, Mimosa spp., Acacia farnesiana and Sesbania grandifora and their affinities with other rhizobia groups. J. Appl. Bacteriol. 49:39- 53.

32. Vincent, J. M. 1970. A manual of the practical study of root- nodule bacteria. International Biological Program handbook 15. Blackwell Scientific Publications, Oxford.

33. Young, J. P. W. 1985. Rhizobium population genetics: enzyme polymorphism in isolates from peas, clover, beans and lucerne grown at the same site. J. Gen. Microbiol. 131:2399-2408.

6 1 :35 1-359.

112:233-238.

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Diversity of Rhizobium Bacteria Isolated from the Root Nodules of Leguminous Trees