4. Characterization of rhizobia nodulating tropical leg um¡ no us trees.

i

I / Abstract

Leguminous trees are abundant in tropical regions of Africa, South America and Asia, where they are valued by foresters and farmers, especially in arid or semi-arid zones, as soil improvers and as sources of firewood and aerial forage. Most tropical leguminous trees form nodules and nitrogen is fixed in symbiosis with rhizobia.

Good characterisation of the rhizobia is necessary to inoculate tree nurseries efficiently. Earlier studies indicated that the bacteria able to form nodules on the roots of tropical trees belonged to the genera Rhizobium and Bradyrltizohiiiti~. Modern taxonomic techniques are pow- erfuf tools that can reveal the great diversity amongst tree rhizobia in tropical areas.They have shown that rhizobia nodulating L ~ I I C N ~ U /eu- cocepyknla aiid related species belong to different known species (Rhizohìuiir loti, R.trc~pici. R.gcrlcgcic, Siiro~-l~i~ohiurn~fr.cc~ìii) and unnamed species (Nií- zobiuiit spp. cmd B~.ci~~~)~.hi=ohiluil spp.). In Senegal we focussed on diverse rhizobia isolated from three Acacia species, A.albida,A.s~.it~.~~~/ and A.fortilis growing in different ecological regions of Senegal. Our results led to the identification of several Bradyrhizobiunr groups nodulating A.albida, three of which are new. Among the fast-growing strains isolated from nodules of diverse Acacia species,we found at least three new groups. One of them represents a new species of the amended genus Sinorhizobium, S. teranga. Another group of fast growers have been differentiated containing at least two new genomic species. Recent results on a great collection of new isolates confirmed definitely the enormous biodiversity of tree rhizobia.

Introduction

In arid and semi-arid tropical areas where severe erosion occurs, trees are O

major importance for maintenance and regeneration of soi1 fertility, cultur yield increase, cattle feeding and production of wood and fruits. In thes respects, tropical leguminous trees play major economical and ecologia roles, because of their ability to fis atmospheric nitrogen in synibiosis wit bacteria belonging to the Rhizobiaceae family. Several authors had earl

~ l l t . aim ofthis communication is to summarize the current taxonomy of the rllizobia, especially those isolated from tropical trees, and to examine the status of rhizobia occuriiig in deep-soil environments.

Taxonomy of tree rhizobia.

F O ~ neiirly a century, all leguine-nodulating bacteria were defined as Rlzizo- b j ~ ~ ~ / ~ ; modern taxonomy using molecular approaches revealed that bacteria inducing nodules on legumes could no longer be put in a single genus. Jordan ( 1) proposed to separate the fdst - and slow - growing rhizobia in two genera, RIiizobiziìn and Bra~vrliizobizulz. RJTizobizinz was divided in three species (2,3): R.Ieg1(i)iiIiosnl.tui.l (coniprising 3 biovars, pliaseoli, trlfolii and viciae), R. meliloti and R loti. Since then two other genera, i. e. Aso~liimbi~iin and Siiiorhizobiunz together with ten species have been proposed (for reviews see 4 and 5). This tendency will increase in the future since heterogeneous groups have been identified already within this classification and more "unknown plants" discovered in the ivorld are investigated for nitrogen-fixing symbiotic bacteria. For example. temperale rhizobia were for a long time the only ones studied but in the last decade several reports on tropical rhizobia have been published (6JS.9.1 O, 1 1 12). Contrary to what was long thought. i.e. tropical soils harboured only wide host-range slowgrowing Bì.N~~~r-Iiizobizrm, these authors showed rhe great diyersity of both fast-and slow-growing nodule-inducing isolates in tropical zones and demonstrated that these belonged to one of four described genera, Rhiso b i m . Bra&rJiko bizmi, Azor-Jiizo b iziriz and Sinorhiso b ium .

ntiI iio\v, taxonomical studies on bacteria nodulating tropical leguminous rees focused essentially on rhizobia associated with two major groups oftrees:

c a e m (Lezicaenrr IezrcocepJiaIr) and related species. and acacias (espe- y sahelian acacias).

27

Rhizobia associated with Leucaena s p p .

The genus Le~rccx17ci Benth. consists 01'50 species: the loctotypc is L ~ I I L " ~ I ? L I leircoceplinin (Lam.)do Wit. \vhich is a troc originating from central Aiiicrica, and is widely used in tropical fbrestry because 01' its high nitrogen-fixing potential. Isolates from nodules ol.' I.. Icucocephala have early bcen reported as Gist-growing rhizobia (13) and to be not spccilic to Lezicneticr and other trees but able to nodulate other leguminous species such as V ì g t ~ i ~ ~ ~ ~ ~ L I ~ c L I I c ~ ~ u ssp. sesqziil,edcilis (snakebenri) ( 14 ). Rhizobia from Lezrccietici leircocey/znlci \,j,'c.re included within the new species R.iofi (2): it \\-as also tlemonstrated ( 15) that \various bacteria capable ofnodulating a n c l fixing nitrogen \vit11 Leiiccimcr Ibrm different DNA homology groups, most probubl>r rcpi-cscntin$ di I'lkrent species. More recenti>'. studying R. Iegrrttiiizoscti.i~irz biovar phnseoli typc 11 strains, Martinez-Romero et NI. (7) proposed (,on the basis o f :I polyphasic a pp ro ac li using ni i l 1 ti 1 oc us enzy ni e e 1 ec t ro p ho re s i s. DNA - D N A li >. b r i d i za t i o n, ribosomal DNA organization, 16s rDNA sequence analysis and phenotypic charateristics) that strains nodulatin,o both Leitccreizcr Leircocepiicrlci sp trees and Phaseolus vulgaris beans, constitute it novel species, R. tropicì. This heterogeneity aning strains that nodulate Lez.ic:ctom species has been recently enlarged by reports ofZhang et al. (8) kom Sudan. md Moreira ef c//. ( 9 ) from Brazil. Leziccretzci isolated fi-om Sudan bclong to four phenotypic clusters and do not group with reference strains (8). Strains isolated from Lezrccreucr species i i17 - - from Amazonian forest and sub-humid tropical forest of the south East c f i Brazil showed a large heterogeneity in their polyacrylamide gel electrophrk- i resis profiles of total cellular proteins (9): some strains group \viti1 a R. loti 4 strain, three of them ivith R-f'redii representatiLTe strains, one with R. gulegae, 3 F and the other form at least four independant clusters. Furthermore, Gao et al. 1 (10) recently confirmed thnt isolates fi-oiii Leircnem spp. from China can If

.

in .$;

met!

Rigi constitute a new group. different from known species.

Rhizobia associated with sahelian acacias. a revc

1 . . ;

P 2 - "-

The genus Acctcicrr Mill is composed ofnumerous species, i.e. 800-900 specï (16), many consisting of trees or shrubs, \videspread in savannas and a regions of Australia. Africa. America and India.

28

between representativc Str¿l¡nS ofthc two clusturs and of'thc dcscribcd R/?izo- bizliiz species lead to the conclus¡on that tach of thc IWO clustcrs observe a status of separate species. One of them was proposcd as a new species of the emended genus Sinor-himbi~iii~. S. tel-nizgcr ( 12). Isolatcs from .acacias origi- nating from North Alkica grouped also in S./ermgu. As the sccond cluster belonged to a large heterogenous one closely rclatccl \vit11 clustcr 3 of Morcira et of. (9) and Lvith at>.pical R. loti strains, we postponed nomcnclaturc decision until more DNA:DNA hybridizations are donc. This \vork is in progress.

All the slow-growinT rhizobia characterized so far around the world have been placed in the genus Bi.ac!l~i.hi,.obiziiiz which appears now as enormous and heterogenous. comprising many clusters. the species status of which are not determined. Only tivo species have been propo,sed to date : B. jcpoi~icziii7 ( I ) , and B. elkciriii (2 I ). Links between all the clusters described rcni~iin to be clone. Eighty-four slo\v-groi\.ing isolates lì-om A.ufDidcr from di ffercnt regions of Senegal. and from diferent ecotypes and depth in soil. were characterized by SDS-PAGE whole-cell proteins analysis (1 I) . All those isolates belonged to the Bi-tríJi-hi_;oDiziiii p ~ i p and they eshibited a great ,dcai of diversit).: 901% of the isolates belonged t o 6 ylectropkoretic clusters. 3 ot' which conraifi reference B,-ad~~r.lii:obizuii strains, B. jnpoiiiciiiii ~ t n d 13. elkcriiii. the other clusters containing only senegalest: isolates. l'lie remaining isolates (1 0%) either group i n 4 small clusters or have separatc positions in the clcndrogram (5 strains). DNA:mRNA hybricii'zations using 16s o r 33S-RNA from B. jnponiczin~ as probe ounfiriiird that all senegalesc il. ci/hid(/ isolates belong ti? the Bradyt-hi_;ohiiitii - /iliollo.~/)ser!~oiilo~~n~ RNA b;-;inch. P irst resid ts 01':

1 GS-rRNA gene sequencing of representatives of 5 gciect!-ophoretic ciusters indicate that 4 of tht'ni constitute a branch as distantl~. separated f'roni B.

jnpoiiicriiii than fi-om .J.fìpici. Blcislobcictei- and Rhoc~o~~~scii~~otii7oiií~s.

rl"

Other tree rhizobia

Rhizobia1 strains isolated í'rom diffcrcn genera of Lropical trees (Prosopis. A 1 b i zzi a. A us t r a I i a I I ;i i' ac i as ) a re o !'ten II c 1 LI d c" CI i n o íi ara c t c r i za t i on stud i es (6,8,9,12, 14,33.33,2.C135):thesc isolates group either ivithin Rhizubizri~r or within Bra&i-blizobiriiiz7 but their exact taxonomical status is rarel). known.

3 o i l

31

+e

+g '-

+s

+e

-I- = nodulating strains; (+) = ineffective nodulating strains;

- - - a = b =

d = e ==.

c =

f = g = h =

y20 nodulation Jarvis et al., (2 ) (polyphasic analysis); Scholla and Elkan, (29) (polyphasic analysis); Martinez-Romero et al., (7) (polyphasic analysis); Zhang et al., (8) (phenotypic analysis); Moreira et al., ( 9 ) (SDS-PAGE analysis); Duptly et al., ( 1 1 ) (SDS-PAGE analysis); de Lajzrdie et al., (1 2 ) (polyphasic analysis); Gao et al., (1 O ) (phenotypic and DNA relatedness analysis).

1

II. Relationships between diversity and spatial distribution.

Desert and arid zones woody legumes are known to develop deep-phreatic roots systems. Significant rhizobial populations exist at the water-level (in thk range of 5.5 to 13 ni) under Prosopis glnndulosa (mesquite) in the Sonor desert (26,27). We observed similar profiles, at a greater depth (34 ni). in Seneg under Acacia albidu (3Q), A.to1stili.s and A.senega1 ( unpublished results).

In Senegal, grouping of isolates from A.rrlbida based on SDS-PAGE anal of the whole-cell proteins was found correlated to geographical ori independant of depth in soil ( I 1 ). On the other hand, in Sonuran Waldon et 1. (23), by nunicrical taxonomic analysis. and Thornas tf al. by ndvB hybridization of PstI-digested genomic DNA. suggested that and deep strains of mesquite rhizobia were clonally related, but that gr strains from each soil depth could be distinguished. They hypothesi the development of distinct rhizobial populations resulted from spatial teniporal separation ofthe surfàce and deep soil that formecl a dry interni soil layer. The differences obscrvod between these two field studies ni due to differences i n the discriminatory power ofthe techniques used, o the various soil conditions. Here again, niorc studies are necessary to un stand the ecologj. of trce rhizobia.

_- 3?

- - a m

9. Moreira FMS, Gillis M, Pot B, Kersters K and Franco AA. 1993 System. Appl. Microbiol. 16 : 135-146.

10. Gao JL, Sun JG, Li Y, Wang ET and Chen WX 1994 Int J. Syst.Bacteriol.44 : 15 1-1 58.

1 1. Dupuy N, Willems A, Pot B, Dewettinck D, Vandendruaene I,. Maestrojuan G Dreyfus B, Kersters K and Gillis M 1994 Int. . Syst. Bacteriol. 44 : 461- 473.

12. De Lajudie P, Willems A, Pot B, Dewettinck, Maestrojuan Gy Neyra M, Collins MD, Dreyfus B, Kersters K and Gillis M 1994 Int. J. Syst. Bacteriol. 44:715-733.

13. Trinick MJ 1968 Exp. Agric. 4 : 243-253.

14. Trinick MJ 1980 J. Appl. Bacteriol. 49 : 39-53.

15. Jarvis BDW 1983 Curr. Microbiol. 8 : 153-158.

16. Allen ON and AllenEK (1980) The Leguminosae - A source Booh of Characteristics; Uses and Nodulation. The University of Wiconsin Press, 8 12pp.

17. Dreyfùs BL and Dornmergues YR 1981 Appl. Environ. Microbio1 41 : 97-99.

18. Dupuy Ny Lorquin J, Ndiaye S, Alasard Dy Gillis M and Dreyfus F 1992 In Plant-Microorganisms Interactions, IFS ed., Stockholm, pp.371-38 1.

19. Gillis M, Pot B, Moreira F, Dupuy N, Dreyfus BL, Maestrojuan G and Kersters K 1992 Conference on taxonomy and automate. identification of bacteria. FEMS Symposium, July 20-24, Prague, Czech os1 ova ki a.

20. De Lajudie P, Lortet G, Neyra M, Badji S, Ndyoe I, Boivin C, Gillis M, and Dreyfbs BL 1992 In Plant Microorganism Interactions, IFS ed., Stockholm, pp.23 8-245.

34

L

2 1. Kuykendall LD, Saxena B, Devine TE and Udell SE 1992 Can. J. Microbiol.3 8 : 50 1-505.

- 22. Lawrie AC 1983 Appl. Environ. Microbiol. 45 : 1822-1828.

23. Waldon HB, Jenkins MB, Virginia RA and Harding EE 1989 Appl: Environ. Microbiol. 55 : 3058-3064.

24. Turk D and Keyser HH 1992 Can J. Microbiol. 38 : 45 1-460.

25. Thomas PM, Golly KF, Zyskind JW and Virginia RA 1994 Appl. Environ. Microbiol. 60 : 1146-1 153.

26. Virginia RA, Jenkins MB and Jarre11 WM 1986 Biol. Fertil. Soils 2 : 127 - 130.

27. Jenkins MB, Virginia RA and Jarre11 WM 1988 Soil Sci. Soc. Am. J. 52 1644-1650.

28. Dupuy NC and Dreyfus BL 1992 Appl. Environ. Microbiol. 58 : 24 1 5-24 1 9.

29. Scholla MH and Elkan GH 1984 Int. Syst. Bacteriol. 34 : 484-486.

Contact address:

M. Neyra, P. delajudie, N.Dupuy and B- Dreyfus. Laboratorie de Microbi- ologie, ORSTOM, B.P. 1386, Dakar, Senegal.

M. Gillis, Laboratorium voor Microbiologie, University Gent, K.L. Lede- ganckstract 35, B-9000 Gent, Belgium.

35

ON

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