Conformity and diversity among field isolates of Rhizobium leguminosarum bv. viciae, bv. trifolii, and bv. phaseoli revealed by DNA hybridization using

chromosome and plasmid probes

GISELE LAGUERRE, ERIC GENIAUX, SYLVIE ISABELLE MAZURIER, RAQUEL RODRIGUEZ CASARTELLI,~ AND NOELLE AMARGER

Znstitut national de la recherche agronomique. Laboratoire de microbiologie des sols, 17, rue Sully, BV 1540, 21034 Dijon CEDEX, France

Received September 16, 1992

Revision received December 8, 1992

Accepted December 15, 1992

LAGUERRE, G., GENIAUX, E., MAZURIER, S. I., RODRIGUEZ CASARTELLI, R., and AMARGER, N. 1993. Conformity and diversity among field isolates of Rhizobium leguminosarum bv. viciae, bv. trifolii, and bv. phaseoli revealed by DNA hybridization using chromosome and plasmid probes. Can. J. Microbiol. 39: 412-419.

A study was made of 113 bacterial isolates from root nodules of peas, lentils, red clover, and French beans, which had been grown in the same soil. Plasmid band profiles visualized in Eckhardt gels were analysed in relation to DNA hybridizationpatterns obtained by probing restricted total cellular DNA in Southern blots. Rhizobium leguminosarum chromosomal probes (placl2, pCOS309.1) and various symbiotic plasmid (nod gene region) probes were used. Dominant plasmid DNA hybridization patterns and more frequent combinations of plasmid patterns and chromosomal types were found among the isolates of each host plant species; the occurrence of alternative combinations indicated that genetic transfer and recombination among members of this soil population had taken place. About 40% of all isolates belonged to the same chromosomal type. Isolates of the same chromosomal type were often found with cryptic plasmids of the same size in different host plant species. Although isolates could not be assigned to their respective plant host groups using chromosomal probes alone, this was generally possible using symbiotic plasmid probes and the results were in complete accordance with plant tests. However, there was a group of bean isolates in which no homology to any of the R. leguminosarum probes was detected under the conditions of high stringency used. Other exceptional isolates of beans conformed in probe tests and subsequent plant host specificity tests better to biovars viciae or trifolii than to biovar phaseoli; thus, the nodulation of beans (i.e., Phaseolus vulgaris) in the field appears less subject to stringent control of specificity than that of other host plant species. It was also noted that the nod gene regions probed showed greater diversity in isolates of biovars viciae and trifolii than in biovar phaseoli.

Key words: Rhizobium leguminosarum, genetic diversity, plasmid, DNA hybridization, restriction fragment length polymorphism.

LAGUERRE, G., GENIAUX, E., MAZURIER, S. I., RODRIGUEZ CASARTELLI, R., et AMARGER, N. 1993. Conformity and diversity among field isolates of Rhizobium leguminosarum bv. viciae, bv. trifolii, and bv. phaseoli revealed by DNA hybridization using chromosome and plasmid probes. Can. J. Microbiol. 39 : 412-419.

Cent treize isolats bactkriens provenant de nodosites de racines de pois, de lentille, de trbfle violet et de haricot, plantes-h6tes cultivkes dans un m&me sol, ont ttC CtudiCs. Des profils de bandes de plasmides obtenus en gel d'Eckhardt ont CtC analyses en relation avec des profils d'hybridation de I'ADN, obtenus par hybridation de I'ADN cellulaire total digere et transfer6 sur filtre selon la mCthode de Southern. Les sondes utilisees proviennent d'ADN chromosomique (placl2, pCOS309.1) et de diffkrents plasmides symbiotiques (regions de gknes nod) de Rhizobium leguminosarum. Des profils dominants d'hybridation des ADN et des combinaisons plus frkquentes de profils d'hybridation plasmi- diques et de types chromosomiques ont kt6 observes parmi les isolats provenant de chacune des plantes-h6tes; I'occur- rence de combinaisons alternatives a indique que des transferts genetiques et des recombinaisons avaient eu lieu entre des membres de cette population du sol. Environ 40% de tous les isolats appartenaient au m&me type chromosomique; les isolats de m&me type chromosomique ont souvent CtC trouves comme ayant des plasmides cryptiques de m&me dimen- sion chez diffkrentes espkces-h6tes. Les rksultats obtenus avec la seule utilisation des sondes chromosomiques n'ont pas permis d'assigner les isolats a leur groupe de plante-hate respectif. En revanche, cette determination a kt6 g6nCrale- ment possible en utilisant les sondes originaires de plasmides symbiotiques et ceci en parfait accord avec les rksultats de tests faits chez les plantes. Cependant, nous avons trouve un groupe d'isolats de haricots pour lequel aucune homologie n'a CtC detectie avec aucune des sondes de R. leguminosarum dans les conditions rigoureuses utilisees. Des isolats excep- tionnels de haricots se sont rCvCles plus conformes aux caracteristiques des biovars viciae ou trifolii qu'a celles du biovar phaseoli d'aprks les tests d'hybridations suivis de tests de nodulation sur plante; ainsi, la nodulation des haricots (c.-a-d., Phaseolus vulgaris) au champ apparait comme moins sujette a un contr6le strict de specificit6 que celle des autres espkces de plantes-h6tes. En outre, nous avons note une plus grande diversite dans l'organisation des regions des genes nod chez les isolats des biovars viciae et trifolii que chez ceux du biovar phaseoli.

Mots elks : Rhizobium leguminosarum, diversite ginetique, plasmides, hybridation de I'ADN, polymorphisme de la taille des fragments de restriction.

[Traduit par la redaction]

' ~ u t h o r to whom all correspondence should be addressed. 'present address: Catedra de Microbiologia, Facultad de Agronomia, Av. Garzon 780, Montevideo, Uruguay.

Printed in Canada / Impnme au Canada

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LAGUERRE ET AL. 413

Introduction Bacteria of the genus Rhizobium form nitrogen-fixing

nodules on roots of leguminous plants. Rhizobium leguminosarum consists of three different biovars (bvs.), namely bv. phaseoli, bv. trifolii, and bv. viciae (Jordan 1984). These biovars are defined in terms of the legume genera they are able to nodulate. For this study it is relevant that bv. viciae nodulates the cross inoculation group of species that includes Lens (lentil), Pisum (pea), and Vicia sativa (vetch), bv. trifolii nodulates Trifolium spp. (clover), and bv. phaseoli nodulates Phaseolus vulgaris (French bean).

Since there is no selective medium for the direct isolation of rhizobia from soil, strains have been isolated almost exclusively from plant nodules. It has been common practice to assign isolates directly to a species and, in the case of R. leguminosarum, to a biovar solely on the basis of the genus of its plant host. In subsequent laboratory tests, how- ever, some isolates have been shown to possess the ability to nodulate alternative hosts (Graham and Parker 1964). It is now known that biovars are determined by plasmid-borne genes that can be transferred among members of the species and may lead to a conversion of specificity from one host t o another (Johnston et al. 1978). Recently, a distinct new species, Rhizobium tropici, has been described that is typically able to form effective nodules with both Leucaena spp. and Phaseolus spp. (Martinez-Romero et al. 1991).

Plant hosts also vary in their specificity towards bacteria. For example, Phaseolus vulgaris is apparently relatively lax; in laboratory conditions, it was found to be nodulated not only by R. tropici and R. leguminosarum bv. phaseoli but also occasionally by the other biovars of R. leguminosarum, other Rhizobium spp., and even Bradyrhizobium spp. However, the nodules produced are often sparse and inef- fective in nitrogen fixation (Graham and Parker 1964; Crow et al. 1981; Sadowsky et al. 1988; Bromfield and Barran 1990; Eardly et al. 1992). Clearly, therefore, for a proper identification or ecological study of bacteria that include R. leguminosarum bv. phaseoli, further information and diagnostic tests are required in addition to the ability t o nodulate beans.

In one ecological study of a natural population of R. leguminosarum isolates, Young (1985) used electro- phoretic type (ET) analysis and reported less diversity among isolates from bean nodules than from clover or pea nodules. Since some ETs were shared by different biovars, diversity was demonstrated at the level of the species and not necessarily at the level of the biovar. This and other studies of diversity in Rhizobium have dealt predominantly with chromosomally encoded characters (Young 1985; Harrison et al. 1987, 1989; Young et al. 1987; Pinero et al. 1988; Eardly et al. 1990). Using restriction fragment length polymorphism (RFLP) analysis, correlations were sometimes found between plasmids and chromosomal types (Schofield et al. 1987; Young and Wexler 1988; Kaijalainen and Lindstrom 1989; Engvild et al. 1990; Demezas et al. 1991 ; Laguerre et al. 1992). Little information is otherwise avail- able concerning the diversity of the plasmids that carry the genes that determine the biovar.

In the present study we provide more information about the genetic diversity of both plasmids and chromosomes, and their assortment in a natural population of R. legumino- sarum. We have used DNA hybridization to analyse bacteria isolated from the nodules of peas, lentils, red clover, and

French beans grown in the same soil. We have thereby aimed to shed more light on the usefulness and limitations of the biovar classification of R. leguminosarum. A study of isolates from peas and lentils formed part of a previous report (Laguerre et al. 1992), which this present work is intended to complement and expand.

Materials and methods Bacterial strains and plasmids

The isolates of R. leguminosarum were previously isolated from root nodules of field plants of pea (Pisum sativum cultivar (cv.) Solara), lentil (Lens esculenta cv. Anicia), and red clover (Trifolium pratense cv. Diper), and of pot plants of French bean (Phaseolus vulgaris cv. Vernandon). The latter were grown in soil collected from the same field as the other legumes and under greenhouse conditions (Mazurier 1989). The sampling site (Institut National de la Recherche Agronomique, Bretennikre, France) had not been cultivated with legumes during the previous 5 years. The initial sample of 476 isolates (approximately 120 per plant species) were categorized by their plasmid profiles, and a representative set of 22,28, 32, and 31 isolates from peas, lentils, red clover, and beans, respectively, was chosen for further study. Isolates were maintained on slopes of mannitol-glutamate agar (Bergersen 1961) sup- plemented with 0.02Vo yeast extract (MGY) at 4OC or in MGY broth with 15% glycerol at - 70°C. Plasmids used in this study are listed in Table 1.

DNA manipulations Plasmid profiles were obtained by agarose gel electrophoresis

using the method of Eckhardt (1978) as modified by Rosenberg et al. (1982). Total DNA was extracted and digested with endonuclease EcoRI and the fragments were separated in 0.9% agarose by gel electrophoresis as previously described (Laguerre et al. 1992). Southern blots were made on nylon filters (Biotrace RP Gelman or Pharmacia LKB Biotechnologie, France) using the Vacugene 2016 apparatus (Pharmacia LKB Biotechnologie, France) according to the manufacturer's instructions. DNA for probes was prepared following alkaline cell lysis and CsCl gradient purification as described by Sambrook et al. (1989). Whole plasmids were used as probes, for vector DNA sequences did not hybridize to DNA from isolates. Nonradioactive DNA labelling with digoxigenin- dUTP, hybridization, and detection were carried out using a kit (Boehringer Mannheim, France) according to the manufacturer's instructions, except that for conditions of high stringency, filters were washed twice for 5 min at room temperature with 2 x SSC (0.30 M NaCl - 0.03 M sodium citrate) - 0.1% sodium dodecyl sulfate (SDS) and twice for 15 min at 68OC with 0.1 x SSC - 0.1% SDS; for lesser stringency, filters were washed twice for 5 min at room temperature with 5 x SSC - 0.1% SDS and twice for 15 min at 68°C with 5 x SSC - 0.1% SDS. The same filters were suc- cessively hybridized with different probes, using the rehybridization procedure recommended by Boehringer Mannheim.

Plant tests Isolates were tested for their ability to form nodules on vetches

(V. sativa cv. Sylphie), red clovers (T. pratense cv. Alpilles), and French beans (Phaseolus vulgaris cv. Vernandon). Seeds were surface sterilized in saturated calcium hypochlorite and germinated on MGY agar plates (red clover) or in sterile perlite (vetches and beans). Seeds of vetch and red clover were grown on slopes of Jensen's agar medium (Vincent 1970). Seeds of beans were grown according to the method of Gibson (1963) using liquid Jensen's medium and filter-paper wicks instead of agar slopes. Inoculation was performed 2 days after planting, using a 1-mL suspension of about 10' rhizobia taken from agar slopes of MGY medium and washed in sterile water. Three replicates were inoculated with each isolate. Plants were grown in a growth chamber (22-20°C day- night temperature; 16-h photoperiod). The root systems were

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CAN. J. MICROBIOL. VOL. 39, 1993

TABLE 1. Plasmids used as probes

Relevant characteristics Reference

pCOS309.1 Cosmid containing chromosomal Cava et al. 1989 LPS gene region (30 kb) of R. leguminosarum bv. phaseoli CFN42; pLAFR1.

pIJ1098 Cosmid containing nodB, C, Lamb et al. 1985 D l , and nolE gene region (ca. Davis and Johnston 1990 20 kb) of R. leguminosarum bv. phaseoli 8002; pLAFRl

pIJ1246 Cloned EcoRI fragment (6.6 kb) Downie et al. 1985 containing nodE, F, D,A, B, C genes of R. leguminosarum bv. viciae 248; pSUP202.

placl2 Cosmid containing chromosomal Young and Wexler 1988 lac gene region (26.2 kb) of R. leguminosarum bv. viciae B155; pLAFR1.

pRt587 Cloned Hind111 fragment Schofield et al. 1984 (14 kb) containing nod genes of R. leguminosarum bv. trijolii ANU843; pBR328.

p42d-7 Cloned BamHI fragment Girard et al. 1991 (4.1 kb) containing nodB gene of R. leguminosarum bv. phaseoli CFN42; pBR329

examined for nodulation at regular intervals. For red clover plants, the effectiveness of the isolates was primarily measured by com- paring plant development with uninoculated controls, but also by nodule and leaf coloration. Effectiveness tests were made on plants of pea (Pisum sativum cv. Solara) and of bean grown in 5-L plastic pots filled with perlite placed on a 5-cm layer of heat-sterilized clay beads. Seeds were surface sterilized as described above, planted eight per pot, and after germination, thinned to four per pot. Plants were grown in a greenhouse under natural light at a temperature not exceeding 2S°C. The nitrogen-free solution (K2HP04, 140 mg/L; MgSO,, 250 mg/L; CaCl,, 280 mg/L; &So4, 120 mg/L; FeCI,, 10 mg/L; H3B03, 2 mg/L; MnSO,, 1.8 mg/L; ZnSO,, 0.2 mg/L; CuSO,, 0.08 mg/L; Na,Mo,, 0.25 mg/L; Co(NO,),, traces) was provided through an automatic watering system. After 7 weeks plants were harvested, shoot dry weight was measured, and the root system was examined.

Results Chromosomal characterization of R. leguminosarum isolates

Total cellular DNA of 113 field isolates of R. legumino- sarum from four host plant species was digested with EcoRI. Southern blots were probed with placl2, which contains chromosomal structural genes encoding P-galactosidase (Table 1). Using highly stringent conditions, no homology to the probe was detected in 11 isolates obtained from beans. However, among the 102 other isolates that did show strong homology to placl2, 22 distinct patterns of hybridization bands were found (for example see Fig. 1). One pattern (al) was common to 44 isolates and detected in 82% (18/22) of isolates from peas, 43% (12/28) from lentils, 19% (6/32) from red clover, and 26% (8/31) from beans. Six of the other hybridization patterns were common to isolates of at least two host plant species from different cross inoculation groups. Each of the 15 remaining pattern types was found in isolates of only one of the four host species (Table 2).

An additional probe, pCOS309.1, carrying chromosomal genes for lipopolysaccharide synthesis, was also used to test isolates from the four host plant species (Table 1). Groupings of isolates giving similar hybridization band patterns with this probe were consistent with those obtained using placl2 (Fig. 1B). Moreover, the 11 isolates from beans that lacked homology to placl2 were also found to hybridize poorly to pCOS309.1 under conditions of high stringency.

An experiment was performed to determine the genomic locations of homology to placl2 and pCOS309.1 in isolates that gave different total DNA hybridization patterns. Prob- ing blots of Eckhardt gels showed that homology to these probes was detectable only in the origin or linearized DNA band and not in any intact supercoiled plasmid band (data not shown). Collectively, these results justified the use of placl2 and pCOS309.1 as specific probes of the R. leguminosarum chromosome.

Plasmid characterization and association with chromosomal types

Plasmid profiles were obtained for all 113 isolates (for examples see Fig. 2). The profiles contained from two to six distinct plasmid bands. Plasmids carrying nod gene regions homologous to those isolated from standard strains of bvs. viciae, trifolii, andphaseoli were identified by prob- ing blots successively with pIJ1246, pRt587, and pIJ1098, respectively (Table 1). The symbiotic plasmids that hybrid- ized ranged in size among the isolates from 180 to 900 kb (Fig. 2). Their size appeared not to be related either to the chromosomal type of an isolate or to the host plant species from which the isolate was obtained. However, it was observed that the size and the number of some nonhybridiz- ing cryptic plasmids were common features of some isolates, and characteristic of the chromosomal type, irrespective of

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LAGUERRE ET AL. 415

TABLE 2. Distribution of chromosomal types among rhizobia isolated from four host plant species

No. of isolates of placl2 pattern typeo Host plant Total of

species ob a1 a2 a2- a3 a3- a4 a4- a5 b cl cl ' c2 d d - e f g h i j k 1 isolates

Pea 18 2 1 1 22 Lentil 12 1 1 3 1 2 4 1 2 1 28 Red clover 6 11 3 2 2 1 4 2 1 32 Bean 1 1 8 1 3 2 1 1 2 1 1 3 1

'EcoRI-digested DNA hybridization patterns with placl2 used as probe as previously described (Laguerre et al. 1992). b ~ o homology detected.

(1) P P L L T T H H Isolate No. 253 115 432 414 115 143 132 4-11

A placl2 hybrldlzatlon

pattern type a1 C1 a1 I a2 c l + a1 I

hybrldlzation

pattern type a ~1 a 1 a ~2 a 1

kb

FIG. 1. Examples of blots with chromosomal probes of EcoRI- digested DNA of R. legurninosarum isolates from the four host plant species. (A) placl2 probe. (B) pCOS309.1 probe. (1) P, pea isolates; L, lentil isolates; T, red clover isolates; H, bean isolates.

the plant host species from which they were obtained (Fig. 2).

The Southern blots of EcoRI-digested total DNA were probed successively with the symbiotic plasmid probes (Fig. 3). With one exception, bean isolates that lacked homology to the chromosomal probes (placl2 and pCOS309.1) failed to show homology to any of the symbiotic plasmid probes under highly stringent conditions; however, weak hybridization was detected under less stringent conditions. Otherwise, greater diversity was observed in the hybridiza-

tion patterns with pIJ1246 and pRt587, derived from stan- dard strains of bv. viciae and bv. trifolii, respectively, than those of p42d-7 derived from a standard strain of bv. phaseoli (Fig. 3A and Table 3). To verify that the paucity of patterns for the latter was not due to the small size of the probe, an alternative bv. phaseoli nod gene region probe, pIJ1098, was used. Although the patterns obtained with pIJ1098 were more complex, the relatively low level of diversity previously found in the bv. phaseoli hybridization patterns was confirmed.

The hybridization band patterns obtained with the sym- biotic plasmid probes suggested a different grouping of the isolates from that obtained using the chromosomal probes (Table 3). More than one type of plasmid was associated with some chromosomal types (e.g., types a l , a2), and conversely, apparently the same pattern was occasionally obtained with widely different chromosomal types (e.g., patterns A and I for pIJ1246) (Fig. 3A and Table 3). It was concluded from these results that, unlike some cryptic plasmids, symbiotic plasmids are not tightly linked to chro- mosomal types in R. leguminosarum.

By contrast, the hybridization patterns obtained with the various symbiotic plasmid probes were found to be generally characteristic of an isolate's host plant species (Fig. 3A). Under highly stringent conditions, homology was usually only detected when the host plant group of the strain giving rise to the probe corresponded with that of isolates under test (Figs. 3A, 3B). However, there were exceptions. Among the bean isolates, there were seven that showed no homology to p42d-7 or pIJ1098, but five of these showed homology to pIJ1246 and two showed homology to pRt587. The five pIJ1246 patterns had already been found among the regular isolates of peas and lentils. However, the two pRt587 patterns were unlike any from the red clover isolates, yet they were both to be found amongst the isolates possessing the predominant chromosomal type (al), which includes isolates of red clover. A further exception was a group of eight isolates, comprising both pea and lentil isolates, which had homology to both pRt587 and pIJ1246 (Fig. 3C). Restriction fragment analysis using pRt587 as probe showed that the plasmid region with homology in the hybrids was less extensive than that found in the regular isolates of red clover.

Bacterial plasmid, chromosome, and host plant specificity Because symbiotic plasmids that were primarily associated

on the basis of nod gene region homology with one host plant species had been found in field nodules of plants of another cross inoculation group, isolates were tested for

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416 CAN. J. MICROBIOL. VOL. 39, 1993

A B C D (1) H P H P T P P T H T H L H L H

Isdate NO. 132 152 344 253 115 256 115 143 225 141 347 231 326 414 441

1 2 3 4 5 1 2 3 4 5 1 2 3 1 2

FIG. 2. Agarose gel electrophoresis of plasmids of R. leguminosarum isolates from the four host plant species. (A) Isolates of placl2 chromosomal hybridization patterns (see Fig. 1) a1 (lanes 1 to 4) and a2 (lane 5). (B) Isolates of placl2 chromosomal hybridization patterns cl (lanes 2 and 4), cl + (lanes 3 and 5) , and c2 (lane 1). (C) Isolates of placl2 chromosomal hybridization pattern f. (D) Isolates of placl2 chromosomal hybridization pattern i. (1) P, pea isolates; L, lentil isolates; T , red clover isolates; H, bean isolates. White symbols localize the Sym plasmids.

TABLE 3. Distribution of symbiotic plasmid (nod gene region) hybridization patterns among chromosomal types of isolates

No. of isolates of indicated nod gene hybridization patternsb with probes:

placl2 pIJ 1246 pRt587 p42d-7 pattern Total no. typea OC A B C D E F G H I J K L M A B C D E F G H A B ofisolates

a1 1 5 8 1 3 5 1 2 3 2 4 34 a2 1 7 3 1 1 13 a2- 3 3 a3 3 3 a3 - 1 1 1 3 a4 2 2 a4- 1 2 3 a5 1 1 b 2 2 cl 2 2 cl + 4 4 c2 1 1 d 1 2 3 d - 1 1 e 2 2 f 2 2 2 6 g 1 1 h 1 1 2 i 1 1 2 j 2 2 k 1 1 1 1 1 od 10 1 11

113

"EcoRI-digested DNA hybridization patterns with placl2 used as probe (Laguerre et al. 1992). b ~ c o ~ ~ - d i g e s t e d DNA hybridization patterns with pIJ1246 (Laguerre et ol. 1992), pRt587, and p47d-7 (see Fig. 3) used as probes. TJo homology detected to any of the nod gene region probes under conditions of high stringency. d ~ o homology detected to placl2 (see Table 2).

nodulation and effectiveness on different host plants. All both pIJ1246 and pRt587, and that were found to nodulate the 55 isolates in which homology to pIJ1246 was detected vetches but not red clover. All other isolates in which (including the exceptional 5 bean isolates) nodulated vetches homology to pRt587 was detected nodulated red clover, (which are a more convenient host in the pea inoculation despite two of them being originally isolated from beans. group for laboratory experiments). This group of isolates Both of the latter and the five bean isolates that showed also included the eight hybrids that showed homology to homology to pIJ1246 were found to have retained the ability

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LAGUERRE ET AL. 417

A I II 111 IV r---------- ir-----r----'l r----'l

(1) P P L L T T H H H H Isolate No. 253 115 432 414 115 143 132 441 132 441

Hybridlzatlon

patterntype A G I A A E A B A1 B

B I II r""""--"--q r-------------

1 (1) P L H H T P L H H T

Isolate No. 253 244 326 441 143 253 244 326 441 143

C I II r------- i r------- 'I

( 1 ) T P L T P L Isolate No. 453 361 342 453 361 342

kb 15 - *

FIG. 3. Examples of blots with nod gene probes of EcoRI- digested DNA from R. Ieguminosarum isolates from the four host plant species. (A) pIJ1246 probe (I), pRt587 probe (11), p42d-7 probe (111), pIJ1098 probe (IV). Hybridizations were performed under stringent conditions. (B) Examples of blots hybridized with pRt587 under stringent conditions (I) and under less stringent conditions (11). (C) Hybrid isolates reacting with both pIJ1246 (I) and pRt587 (11) under stringent conditions. (1) P, pea isolates; L, lentil isolates; T, red clover isolates; H, bean, isolates.

to form nodules on beans. However, these nodules were symbiotically ineffective, being either few in number or delayed in appearance, yet they were effective in nitrogen

fixation on the host plants with which the probes were normally associated. The ability to nodulate beans, albeit ineffectively, was also found in six of a sample of seven typical isolates from peas and lentils that showed regular bv. viciae homology to pIJ1246, and also in those excep- tional bean isolates that showed no homology to the chro- mosomal probes. In this study, symbiotically effective nodules were formed on beans only by isolates that showed homology to p42d-7 or pIJ1098.

These results demonstrate the potential usefulness, and limitations, of probes derived from the nod gene regions of different R. leguminosarum biovars for the characterization of field isolates.

Discussion DNA hybridization using chromosomal sequences as

probes has been previously used to differentiate between R. leguminosarum isolates (Schofield et al. 1987; Young and Wexler 1988; Demezas et al. 1991; Segovia et al. 1991; Laguerre et al. 1992). We have used two such probes, placl2 and pCOS309.1, which we first verified did not hybridize to plasmids in a test sample of isolates. This precaution was deemed to be necessary because a P-galactosidase was found to be plasmid encoded in Rhizobium meliloti (Charles et al. 1990) and lipopolysaccharide synthesis genes had been found to be present on plasmids of R. Ieguminosarum (Hynes and McGregor 1990; Baldani et al. 1992; Brom et al. 1992). We then examined nodule isolates from peas, lentils, red clover, and French beans, which were members of the same field population.

We discovered that about 30% of the bean (Phaseolus vulgaris) isolates possessed no detectable homology to either chromosomal probe. Evidence supporting the classification of this group as a species distinct from R. leguminosarum and R. tropici will be given elsewhere. There have been previous reports that the bacteria that nodulate Phaseolus vulgaris constitute a heterogenous group of species (Crow et al. 198 1 ; Pinero et al. 1988; Martinez-Romero et al. 1991 ; Eardly et al. 1992).

Among the other isolates, we found a variety of hybrid- ization patterns of EcoRI restriction fragments using the chromosomal probes. The same pattern types were often found in isolates of different host plant species such that we were unable to distinguish biovars of R. leguminosarum using these probes alone. This result is consistent with the finding that R. leguminosarum isolates from peas, white clover, and beans were not distinguishable as biovars solely by enzyme polymorphism, which is largely an expression of chromosomal differences (Young 1985). The most fre- quently occurring hybridization pattern (al) was dominant in all groups of isolates except those from red clover. This is in contrast to the finding of Hynes and O'Connell(1990) who reported different dominant types even among isolates of the same inoculation group of host plants. In particular, they observed a striking difference between isolates of peas and faba beans whereas we detected no such difference between isolates of peas and lentils. Nevertheless, lentils did give isolates with a wider range of chromosomal pattern types than peas, and this may well reflect a difference in the degree of host specificity.

Among the R. leguminosarum isolates of the present study, there was great diversity in the size and number of plasmids present in the genome. Some cryptic plasmids appeared to be highly conserved and often associated with

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418 CAN. J. MICROBIOL. VOL. 39, 1993

a chromosomal type, independently of the isolates' host plant species. Symbiotic plasmids, which were very variable in size, were categorized by probing with various nod gene region probes. By contrast, these were clearly associated with particular plant hosts. The probes used were nod gene region clones derived from standard strains of R. leguminosarum biovars (viciae, trijolii, and phaseoli) proven in their sym- biotic effectiveness. Considering that they all contained nod structural genes, it was perhaps surprising how specific was the hybridization, albeit under conditions of high stringency. No bacteria were isolated from nodules of peas, lentils, or red clover or successfully used to nodulate these plant hosts (or plant species of their cross inoculation group) in the laboratory, which did not have homology to probes specif- ically associated with the cross inoculation group of that host plant species.

Beans are considered here as a separate category because our results showed that nodules were formed with a wide range of bacteria. This clearly demonstrates that the pro- miscuity observed in bean plant nodules in laboratory conditions also occurs in field situation where there is com- petition between rhizobia. We found bean isolates that con- tained homology to the bv. viciae or bv. trijolii probes in addition to those isolates with regular homology to the bv. phaseoli probes. We also found isolates with homology to none of these plasmid probes, at least under conditions of high stringency. Only~isolates with homology to the bv. phaseoli probes were able to nodulate beans effectively in terms of nitrogen fixation.

The nod gene region probes revealed an interesting anomaly among the pea and lentil isolates. There were eight whose specificity for host plants, when taken with the results of the bv. viciae plasmid specific probe, indicated that they were typical of bv. viciae. However, further probing showed that they had additional homology to the bv. trijolii probe. None of these 'hybrids' formed nodules on red clover.

In general, the nod gene region sequences having homology to the host-specific plasmid probes of bv. viciae and bv. trijolii appeared more variable in EcoRI digests than did the chromosomal regions. Dominant plasmid hybrid- ization patterns could be recognized and thus were fre- quently associated with dominant chromosomal types. This may be a chance phenomenon, and not an expression of preference, although the finding is consistent with previous reports (Engvild et al. 1990; Demezas et al. 1991). However, the dominant plasmid patterns were also found to be associated with different chromosomal types, and vice versa, which also confirms earlier reports and suggests plasmid transfer events (Schofield et al. 1987; Young and Wexler 1988; Laguerre et al. 1992). The distribution of symbiotic plasmids across the chromosomal types could be much wider than that observed within a sample of rhizobia isolated through the host plant. Again the situation in beans is different, for little variation was found in the nod gene region of isolates having homology to bv. phaseoli probes. It may be more than coincidence that P. vulgaris is a relatively recent introduction to agriculture in Europe.

The results presented here allow the following conclusions concerning the analysis of field populations of R. legumino- sarum. Chromosomal probes are useful for the analysis of general diversity within the species. Collectively, chromo- somal and nod gene region probes can resolve the intricate tripartite relationships of host plants, bacterial (chromo-

somal) types, and symbiotic plasmids. We have demon- strated dominant combinations of, and recombination between, these three elements in our study of one field population. The classification and tenure of a strain as a biovar are largely dependent on the presence and stability of the least stable element, namely the symbiotic plasmid (Soberon-Chavez et al. 1986; Flores et al. 1988; Romero et al. 1991). Insofar as the nod gene region probes specifi- cally identify the groups of primary plant hosts of isolates (via their symbiotic plasmids), they are likely to be useful for the direct isolation, screening, and study of natural populations of R. leguminosarum, independently of plant nodulation and selection. It is then hoped that genetic factors affecting these important events in the field will be identifiable.

Acknowledgements We are very grateful to R. Wheatcroft for his critical

discussion and help with improving the manuscript. We wish to thank M.R. Allard for technical assistance, E. Martinez- Romero and J.P.W. Young for supplying us with strains, E. Martinez-Romero for providing p42d-7 and pCOS309.1 probes, J.M. Watson for providing pRt654 and pRt587, and J.P.W. Young for providing placl2, pIJ1246, and pIJ1098. We also thank N. Parekh for helping with the English text.

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