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Home , Bradyrhizobium, Rhodopseudomonas

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JlREyFus 64 - 9c2 DIVERSITY OF PHOTOSYNTHETIC STRAINS FROM STEM NODULES OF

*, J. LORQUIN, F. MOLOUBA, N. DUPUY, S. NDIAYE, D. ALAZARD, M. GILLIS AND B. DREYFUS 'LABORATOIRE DE MICROBIOLOGIE, ORSTOM, BP. i386 DAKAR, SENEGAL 2~~~~~~~~~~~ VOOR MICROBIOLOGIE, UNIVERSITEIT GENT, K.L. . LEDEGANCKSTRAAT, 35, B-9000, GHENT, BELGIUM

INTRODUCTION Tropical of the genera Sesbania and Aeschynomene are known to form nitrogen-fixing nodules on their stems. Because of their high N,- fixing potential, these plants are successfully used as green manure in paddy-fields and significantly improve the rice yield (Ladha et al. , 1988; Ndoye, 1990). In both genera, nodule formation occurs at predetermined sites which always correspond to root primordia present all along the stem (Dreyfus and Dommergues, 1981). Sesbania rostrata is known to be associated with both and Azorhizobium, the latter genus being very specific to this species (Dreyfus et al., 1988) - In the genus Aeschynomene which comprises about 250 tropical species, more than 20 species are known to bear stem-nodules. According to their nodulation, Aeschynomene species are divided into three groups : in the first group (eg. Ae. americana, Ae. elaphroxylon) plants are only nodulated on roots or submerged part of the stem. The second (eg. Ae. afraspera, Ae. nilotica) and third group (eg. Ae. indica, Ae. sensitiva) comprise all stem-nodulating species. They differ by cross-inoculation pattexns and by the accessibility to infection of the root primordia present on the stem, the second group being more profusely nodulated than the third group (Alazard, 1985 ; Alazard and Duhoux, 1988). The first group of Aeschynomene is known to be nodulated by typical slow-growing strains of the genus Bradyrhizobium (Alazard, 1985). Until recently, this heterogeneous genus which includes all slow-growing strains has not been divided into species, except that strains nodulating (Glycine max) are conventionally called . At least three DNA homology 653 R Palncios er al. (e&.), New Horizons in Nitroger1 Fi.raiiori, 683-689. 0 I993 Kliiwer Academic Piiblisliers. Printed Li the Nerherinrzds.

Fonds Documentaire ORSTOM 1 1 il,

684 groups have been differentiated (Hollis et al., 1981). Recently, Kuykendall et a1.(1992) proposed a new species for members of the DNA homolgy group II. Bradyrhizobium japonicum is closely related to the purple non-sulfur photosynthetic bacterium palustris (Young et al., 1991) and to Blastobacter denitrificans and the genera and Nitrobacter . (Willems and Collins, 1992 ; Young, 1992). Moreover, the slow-growing strain BTAil isolated from stem nodules of (group III) was shown to be a photosynthetic bacterium (Evans et al., 1990). Although BTAil had physiological and growth properties that were considered more characteristic of Rhizobium than that of Bradyrhizobium, it was also recently shown to belong to the Bradyrhizobium- Rhodopseudomonas rRNA cluster (Young et al., 1991). We have recently isolated a large collection of photosynthetic and non photosynthetic strains from stem-nodules of several species belonging to the three groups of Aeschynomene. The aim of this work was to determine by taxonomical studies whether all these strains were related to the Bradyrhizobium genus. Furthermore, we have described on the stem of Aeschynomene sensitiva a new type of nitrogen-fixing nodule formed under the stem epidermis by photosynthetic that we named stem cbllar nodule.

NODULATION SPECIFICITY AND PHOTOSYNTHETIC BRADY RH I ZOBIA IN AESCH YNOMENE We isolated 126 strains o€ bradyrhizobia from stem nodules of Aeschynomene americana, Ae. elaphroxylon, Ae. pfundii, Ae. schimperi, Ae. uniflora, Ae. afraspera , Ae. nilotica , Ae. ciliata, Ae. indica , Ae. sensitiva and Ae. tambacoudensis. Among them, we observed that 83 strains synthetized coloured pigments when grown aerobically in Yeast- Mannitol solid and liauid culture medium (Vincent. 1970) under intermitter& incandescent light '( 14 hours light, 8 hours dark). They were found to contain bacteriochlorophyll a (Bchla) in a protein-associated complex (hmax 800 and 870 nm) and carotenoids with zharacteristic spectra (between 300 and 550nm). Unlike photosynthetic non-sulfur bacteria belonging to the closely related genus Rhodopseudomonas, bradyrhizobia produced carotenoids and Bchla only under strictly aerobic conditions and failed to grow anaerobically under photoautotrophic 32 photoheterotrophic conditions. Three types of photosynthetic strains were observed according to . their culture pigmentation, the light pink strains (LP) similar to BTAil, the dark pink strains (DP) and the orange strains (O). Forty three strains remained white (W) under light and no carotenoids could be - detected after extraction and spectroscopic analysis - (Table 1). The 126 photosynthetic and non-photosynthetic strains were then tested for nodulation and nitrogen - fixation on different Aeschynomene species belonging to the three groups of Aeschynomene. Results are summarized in Table 1 and confirm the division of Aeschynomene species into three cross-inoculation groups. Non-photosynthetic strains were found in groups I and II, but were effectively fixing nitrogen in group 1 species only. Photosynthetic strains are effectively nodulating group II and III, this last group being more specific with a narrow host-range. Photosynthetic strains never formed nodules on Aeschynomene species of group I. In group II, photosynthetic strains appeared to be the most effective. OF AESCHYNOMENE BRADYRHIZOBIA isolated from Aeschynomene were characterized by a comparative study of total proteins using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic separation of whole-cell proteins have been widely used in Rhizobium, Azorhizobium and Bradyrhizobium taxonomic studies (Kersters, 1985 ; Moreira et al., 1992). This discriminative and reproducible technique provides information at the level of species and subspecies. The bacterial protein patterns of some photosynthetic and non-photosynthetic Aeschynomene bradyrhizobia were scanned and analysed numerically together with the data of patterns of reference strains available in the data base of our research group at the University of Ghent (Belgium). Preliminar results summarized in Fiqure 1 showed that strains isolated from Aeschynomene all belong to the Bradyrhizobium group and are members of different gel electrophoretic clusters : the non-photosynthetic strains belong in different known subclusters, while the photosynthetic ones seem to constitue three new subclusters. A more profound evaluation of these results is necessary in order to definitely confirm the existence of these new subclusters. Because the carotenoids content of all strains has not yet been determined, it is not possible to decide whether ,.

o\ Table 1. Cross-inoculation specificities in the genus Aeschynomene. 03 íh

Cross - Test plant* inoculation Bchlal -pattern Bacterial Strain and Ae.elaphroxy1on Ae. afraspera Ae.indica Ae. sensitiva strain color' carot. Host plant Group I Group II Group III

Re. afraspera ORS 285 LP + O E E E ORS 362 LP + O E E E Group III Ae. sensitiva ORS 276 LP + O O hE hE Re. indica ORS 399 LP + O O E E

Ae. indica ORS 344 DP + O O E E ORS 371 DP + O O E E

Re. sensitiva ORS 278 O + O O E hE ORS 279 O + O O E hE

Culture grown in a YM medium under intermittent light. W, White ; L P, Light pink ; D P, Dark Pink ; O, Orange. O, no nodules produced. hE, E, e and I respectively high effective, effective, partially effective and ineffective root nodulation. 6 I'

('00 1.95 1.90 g.85

Photosynthetic Bradyrhizobium sp. Level of similarity ( Aes ch yn omen e ) ( new c 1us ter including BTAil)

Photosynthetic Bradyrhizobium sp. (Aeschynomene) (new cluster)

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Rhizobiumi

Figure 1. Simplified dendrogram showing the SDS-PAGE relationships ' among Bradyrhizobium sp. nodulating Aeschynomene sp., based on r values as calculated by the unweighted method using average 03 linkage. .I 3 .. 688 these new clusters correspond respectively to the pink, dark pink and orange coloured strains. Furthermore, DNA from a few representative strains of Aeschynomene were tested for DNA : rRNA hybridization with a labelled rRNA probe from Sradyrhizobium japonicum NZP554gT. The results ionfirmed that they belong to the Bradyrhizobium rRNA Auster.

STEM COLLAR NODULE IN AESCHYNOMENE SENSITIVA As in other Aeschynomene species, stem infection by Bradyrhizobium strains in Ae. sensitiva always occurs at predetermined sites which correspond to root primordia present just beneath the stem epidermal cell layer. Seven days after inoculation by the photosynthetic strain ORS 278 an enlargement of the root primordia is macroscopically observed simultaneously when begins. From the root primordia, infection rapidly seems to progress in the stem cortex. Continuous horizontal stem meristematic activity then forms a collar all iround the stem. Later, the :ollar becomes enlarged and a senescent zone appears in its middle. Finally two collar nodules are formed )n each side of the senescent central zone. A :ytological study of this new type of nodule is being )erformed in our laboratory. !ONCLUSIONS

We have shown that the ability to form iacteriochlorophyll a and photosynthetic reaction !enters was a common feature to most bradyrhizobial trains isolated from stem-nodulated species of eschynomene. These photosynthetic strains formed hree protein gel electrophoretic clusters, of which he mutual relationships and the relationships with he other Bradyrhizobium subclusters has to be nraveled further. Diversity of the strains was onfirmed by their carotenoid content. Furthermore, a new stem collar nodule was escribed which allows the same light intensity for 11 infected cells. Perhaps such aerial nodule tructure could explain how, during evolution, hotosynthetic capability of the rhizobia have been itained in the symbiotic association. I n t ere s t ing 1y, photo synthetic Bradyrhizobium trains appeared the most effective for nodulation nd nitrogen fixation of the stem-nodulated species Ich as Aeschynoneme afraspera which, like Sesbania #a 2-i -- Y

rostrata, is widely used as green manure in paddy- fields. Studies under way in our laboratory should determine whether this higher nitrogen fixing activity is due to the close association of and N,-f ixation.

REFERENCES

Alazard D (1985) Appl. Env. Microbiol. 50, 732-734. Alazard D and Duhoux E (1988) J. Plant Physiol. 132, 123-125. Dreyfus BL and Dommergues YR (1981) FEMS Microbiol. Lett. 10, 313-317. Dreyfus B et al (1988) Int. J. Syst. Bacteriol. 38, 89-98 Evans WR et al (1990) Appl. Environ. Microbiol. 56, 3445-3449. Hollis AB et al (1981) J. Gen. Microbiol. 123, 215-222. Kersters K (1985) In Society for General Microbiology, eds, Computer-assisted bacterial systematics. Kuykendall LD et al (1992) Can. J. Microbiol. 38, 501-505. Ladha JK et al (1988) In The International Rice research Institute, eds, Green manure in rice farming, pp 165-183 , Manila. Moreira F et al (1992) In press. Ndoye I (1990) PHD, Universite de Lille, pp 103-129. Vincent JM (1970) A manual for the pratica1 study of bacteria, Blackwell Scientific Publications, Oxford. Willems A and Collins MD (1992) FEMS Microbiology Letters 96, 241-246. Young JPW et al (1991) J. Bacteriol. 173, 2271-2277. Young JPW (1992) In Biological Nitrogen Fixation, 43-86, Chapman and Hall, New York, London.

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