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transferred to R. leguminosarum biovar phaseoli RCR 3644-S1. The transconjugant selection had been done by Trifolium pratense plants. All transconjugants ...
CURRENT MICROBIOLOGY Vol. 19 (1989), pp. 179-181

Current Microbiology 9 Springer-Verlag New York Inc. 1989

Selection and Symbiotic Properties of Rhizobium leguminosarum Biovar phaseoli Strains Harboring pRtr5a M. Rosario Espuny, F. Javier Ollero, and Ramdn A. Bellogfn Department of Microbiology, Faculty of Biology, Facultad de Biologfa, University of Seville, Seville, Spain

Abstract. The Rh&obium leguminosarum biovar trifolii symbiotic plasmid pRtr5a has been transferred to R. leguminosarum biovar phaseoli RCR 3644-S1. The transconjugant selection had been done by Trifolium pratense plants. All transconjugants lacked the resident pSym, but had complete pRtr5a, and were Fix + on T. repens and T. alexandrinum, Fix- on T. subterraneum, and formed a few small white and Fix- nodules on Phaseolus vulgaris. It is shown that this nodulation on P. vulgaris is due to pRtr5a. The presence of pRtr5a and/or the passage through Trifolium pratense nodules provoke(s) the recipient strain symbiotic plasmid loss.

Bacteria belonging to the genus Rhizobium fix nitrogen symbiotically with leguminous plants. Rhizobia infect leguminous roots and develop certain structures, named nodules, inside which the molecular nitrogen fixation process is carried out. In the symbiosis establishment, bacterium as well as plant genes are involved, and complicated interactions, not entirely known, occur. But not just any Rhizobium can infect a particular leguminous plant because of the high specificity of the process. The host range, in addition to nodulation and nitrogen-fixation abilities, is determined by genes, which are harbored on plasmids in most of the Rhizobium strains [3, 11, 13], called symbiotic plasmids (pSym). The transfer of pSym from one Rhizobium species to another leads, in most cases, to recipients capable of nodulating host plants of both the donor and recipient. In other occasions the transfer of the pSym between species does not determine the establishment of effective symbiosis on the new host plants [1, 14] in spite of the two symbiotic plasmids being complete. Frequently incompatibility phenomena appear, giving rise to the loss of one of the Sym plasmids [9]. The object of this paper was to study the Sym plasmid pRtr5a (which harbors genes for nodulation of Trifolium) expression on Rhizobium leguminosarum biovar phaseoli strain and the possible incompatibilities with resident plasmids.

Materials and Methods Microbiological methods. Bacterial strains and plasmids are shown in Table 1. Media were described previously [9], The antibiotic-resistant strains were obtained as described previously [9]. The melanin production was determined by the method described by Cubo et al. [8]. Bacterial matings were carried out according to the method described by Buchanan-Wollaston et al. [6]. In other cases the transconjugants were obtained according to the select plant method [4]. To determine the pRtr5a stability in transconjugants, we used the method described by Wang et al. [16].

Plasmid visualization. Plasmid visualization was carried out by electrophoresis in agarose gel according to the method described previously [9].

Nodulation tests. The symbiotic properties of the different strains were tested on Trifolium repens, T. pratense, T. atexandrinum, T. subterraneum, and Phaseotus vufgaris. The method followed with Trifolium seeds has been previously described [9]. The P. vulgaris seeds were also surface-disinfected by inmersion in sodium hypochlorite (12%, wt/vol) for 15 min; then they were washed 10 times in sterile water before being placed on TY plates in order to determine the effectiveness of treatment. TY plates were kept in the dark at 20~ for seed germination. The seedlings were placed into Erlenmeyer flasks containing vermiculite and supplemented with Fahraeus nutritive solution [ 15], pH 6.8. The flasks, with one plant each, were kept in the dark at 20~ for a week, then inoculated with 5 ml of the rhizobia suspension. After inoculation, the Trifolium and P. vulgaris plants were grown as described previously [9]. The plant roots (4-6 weeks old) were cut off, and nitrogen fixation was measured by the acetylene reduction technique.

Address reprint requests to: Dr. M. Rosario Espuny, Departamento de Microbiologfa, Facultad de Biologfa, Apdo. 1095, 41080 Sevilla, Spain.

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CURRENT MICROBIOLOGY Vol. 19 (1989)

A

Table 1. Bacterial strains and plasmids Strain

Relevant properties a

B

C

D

E

F

G

Reference

R. leguminosarum biovar phaseoli RCR 3644

Wild isolate; melanin producer; also called 1233 RCR 3644-S1 Spontaneous sm-r derivative of RCR 3644 REP-1 to REP-16 From cross AB 148 x RCR 3644-S 1 R. leguminosarum biovar trifolii RS169.2NA545 Nod- by curing of pSym derivative of RS169 RS 169.NA3 Spontaneous rif-r derivative of RS 169.2NA545 RS169.NA3.phl From cross REP-1 x and 2 RS 169.NA3

[12] This study

[2] This study This study

Rhizobium fredii AB 148

Fast-growing USDA 194.rifr (pRtr5a) R. teguminosarum biovar viceae T3 ura-14, trp-16, str-86 pJB5Jl

[7]

[10]

Plasmid

Markers carried

Reference

pRtrSa

Tra +, Nod +, Fix + (on Trifolium), Tn5

[11]

a Abbreviations: rif-r, resistance to rifampicin; sin-r, resistance to streptomycin; Tra +, conjugative; Nod +, ability to form nodules; Fix +, ability to fix nitrogen within nodules.

Isolation of Rhizobium from nodules. Nodulated plants, before

flowering, were taken and the nodules cut off, then surface-disinfected as previously described [9].

Results and Discussion The recipient strain was a spontaneous streptomycin-resistant mutant, obtained at a frequency of 10-8, melanin producer, and Fix + on Phaseolus vulgaris. As can be seen in Fig. 1, this strain showed three bands (of about 310, 230, and I90 Md). The smallest band (190 Md) corresponded to the pSym [3]. The pRtr5a donor was the strain AB 148 of Rhizobiumfredii, which was unable to nodulate on Trifolium in spite of harboring complete pRtr5a. This strain, in addition to pRtr5a (180 Md), has a plasmid of more than 600 Md (Fig. 1). As it was not possible to transfer pRtr5a by the Buchanan-Wollaston method, transconjugants were selected by T. pratense plants, taking advantage of the fact that neither donor nor recipient nodulate on Trifolium.

Fig. 1. Plasmid profiles in strains: A, T3; B, RCR 3644-$1; C and D, transconjugants from cross AB 148 x RCR 3644-S1; E, AB 148; F, RS169.NA3.phl; and G, RS169.NA3. The plasmid sizes are rough approximations obtained by comparison with plasmids from Rhizobium leguminosarum biovar viceae strain T3. The molecular weights of the se plasmids are as follows: 310,285,220, 165, 130, 100, and 90 Md [5].

Sixteen transconjugants were taken from sixteen different nitrogen-fixing nodules. All of them were non-melanin-producing and showed the same electrophoretic profile, consisting of the recipient 310 and 230 Md plasmids and, instead of the 190 Md plasmid (pSym), they showed one of 180 Md (Fig. 1). Beynon et al. [3] have reported that the RCR 3644-S1 pSym is deleted until 180 Md, and the strain harboring this plasmid was mel- and Nod- on P. vulgaris. This type of deleted plasmid also has been spontaneously obtained during this work. So it is not possible to know whether the transconjugant strains have lacked this pSym or whether it is deleted to 180 Md, coinciding with the electrophoretic band corresponding to pRtr5a (180 Md). All transconjugants presented the same symbiotic properties: Fix + on T. repense and T. alexandrinum, Fix- on T. subterraneum, and formed from one to five white, small Fix- nodules on P. vulgaris like the donor AB148. The inability to fix nitrogen on T. subterraneum could mean that a different fixation control mechanism on this species exists. All T. repens and T. alexandrinum nodules isolates were km-r except REP-2 when isolated from T. alexandrinum; in this case, one of the ten colonies examined was km-s. However, when some transconjugants were isolated from T. subterraneum or P. vulgaris nodules, both km-s and km-r colonies were obtained. The kanamycin resistance stability was determined in the transconjugant REP-12; only one of the 884 colonies examined was km-s (spontaneous lost frequency of about 0.1%). The transconjugant plasmid profiles after the

M.R. Espuny et al.: Symbiotic Properties of R. leguminosarum Biovar phaseoli (pRtr5a)

passage through plant nodules were maintained in all cases, except when km-s colonies were isolated, which lacked the 180 Md band (pRtr5a). The km-s isolates were Nod- on the four Trifolium species used as well as on P. vulgaris, indicative that the transconjugants did not contain the deleted RCR 3644-$1 pSym (180 Md), suggestive of the incompatibility between pRtr5a and the recipient pSym. The possibility cannot be discarded that both plasmids could coexist in the same recipient cell and the Trifolium plants exert some type of contraselection over the RCR 3644-$1 pSym. On the other hand, the results suggest that the nodulation of transconjugants on P. vulgaris is due to the presence of pRtr5a. To confirm this, pRtr5a was transferred from REP-1 to Rhizobium leguminosarum biovar trifolii RS169.NA3 (Nod- by curing of pSym). The transfer frequency was about 10 -6 per recipient cell. Two transconjugants (RS 169.NA3.phl and 2) were taken; they were Fix + on T. pratense, and the nodule isolates maintained the km-r marker. Likewise, their plasmid profiles showed a new 180 Md band (pRtr5a) in respect to RS169.NA3 (Fig. 1). In the nodulation test on P. vulgaris, harboring pRtr5a strains (AB 148 and RS169.NA3.ph1) and non-harboring pRtr5a strains (km-s REP-5 and REP-15, obtained after the passage through P. vulgaris nodules, and km-s REP-2 obtained after the passage through T. alexandrinum nodules) were used as inoculants. Once again, one to six small white and Fix- nodules were produced when the plants were inoculated with the harboring pRtr5a strains, while the strains without pRtr5a were Nod-. Thus, pRtr5a is capable of inducing this erroneous nodulation on P. vulgaris. Literature Cited 1. Appelbaum ER, McLoughlin TJ, O'Connell M, Chartrain N (1985) Expression of symbiotic genes of Rh&obium japonicum USDA 191 in other rhizobia. J Bacteriol 163:385-388 2. Bellogfn RA, Espuny MR, Guit6rrez-Navarro AM, P6rezSilva J (1984) Polysaccharides and lipopolysaccharides and infectivity ofRhizobium trifolii. Soil Biol Biochem 16:23-26

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