On the othcr hand. nize that the present genelic based. in many cases, on the vast genctic intbrrnatiorr rsm. To make the scope ...... rn the spccies Bortletellu R. p?rtussis (see'fable 2) purapertu.ssi.s and ..... Grosskinsky, C. \1., Jacobs, lV. It.. .1r.
( ' r i t u l R e v i e r titn P [ a n tS c i t n c e sl,5 ( 2 ) : l l - l 1 1 0( 1 9 9 6 )
RhizobiumPhylogeniesand Bacterial GeneticDiversity EsperanzaMartfnez-Romero* and Jesils Cabaltero-Mettado Departamentode Gen6ticaMolecular,Centro de Investigacionsobre Fijacronde N i t r 6 g e n oU, N A M .A P 5 6 5 - A , C u e r n a v a c aM, o r e l o sM , 6 x i c oF ; a x7 3 - 1 7 - 5 5 - 8 1 ; T e l ( 7 3 ) - 1 31-6 - 9 7 e ; m a i l= e m a r t i n @ e c i fn .u n a m . m x Fleleree: Dr.JanetSprent,Department of BiologicalSciences, universityof Dundee,DundeeODl 4HN, Scotland,U.K. i
Corresponding author. AIISTRACT: The l-egunrinosae is oneoI rhelargestianriliesof planrs.It hasa broadgeographi cal distribution.The principal)cgurnespccieshavedefincdsitcsoforigin andthesccoincidewith thedivcrsit'ication centcrstbr thcir "spc'cific symbioticbacteria.Thesenitrogenfixin-qbacteria. which forrn nodulesin the roots or stcmsof the plants,bclong to dilterent bactcriallineages tRlri:.biur,, Bntdtrlti:t >iunr,ond A:orhizobiun)rclated to other nonsymbioticbacterir. A remarkablechilracteristic of thesebacteriais their largegeneticdiversity.The genericrelation shrpsamongthc differenrbactcrialgroupsarebcingdeliDcdbasednrainl\ on thc analysisof the sequcrcesof the ribosomalgenes.Rccentresultspoinl our the needto havea broaclergcnonrrc scopc.Genc maps,genomesizes.and sequence ol metabolicgenes\\'oulci\en,e to Validatcthe presentRhi:.obium,tnd Bradtrhi.ohiumphylogenics.N,lorerealisticphylogeniesshouldpcrhaps considerlateraltransftr bet\r,ccnclustersof bacteria. A compilationof recordsof bacterialgeneticdiversity.includingentcrobacteria and pathogens.is presentedand comparcdwith Rhi:obiundiversitl'.It is proposedthat humanacriviries are having importanteffcctson nicrobe diversitv. KEY IVORDS: Rhizobium,taxonomy,phylogenies. diversity.parhogens. enterobacrcria.
I. RHIZOBIUM PHYLOGENIES A. Introduction The Leguminosae compriseone of the largestfamiliesof plantswith over I 7,000to 19.000describedspecies.a It is estimated that the differentspecieswere alreadydivergingby the time the continentalmasses were separatlngapproxlmately6-5million yearsago.tr6.tr8.lr0 Differentlegumesevolved and diversifiedin various regionsof the world.For example,the soybeandeveloped
in Asia;l{)7 Phaseolusspcciesllourishedin bothMesoamerica andin theAndeanrcgion of South America:72 Trifoliun, Medicago, andPistunspeciesin the Middle Eastr:r'rrl andGalegaoJJicinalisandG. orientulisproltably in the Caucasus.r15 Afiica is the only siteof origin of bambaragroundnut(Vigrra subterranea'1, cowpea(V. ungui(Ltlata). and ( t l h e r \1 ar s r l n r d d i t i 6 n t o l h e \ c m t i n c r ( ' p \ .
perhapshundredsof legumcsalsohavespecific drversification sites.r00 Other,perliaps older legumes,suchas Acacla.with about 1500species, aredistributed worldwide.r5L
0715,2689/96/$.50 O 1996bv CRCPress. Inc.
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It is generally consideredthat the Legunr inosae family has both a tropical and subt r o p i c a l o r i g r n a n d t h a t c o l o n i z a t i o na n d a d a p t a t i o nt o t c m p l a t e r e g i o n s o c c u r r e d later.iE Legumes are rer.narkablebecausethey e s t a b l i s ha s y m b i o t i ci n t e r a c t i o nw i t h n i t r o gen-fixrng bacteriafrom the generaRftl:obiunt, Bradyrhi:obium, and A:orhi:obiun. which form noduleson the ru)ts or stemsof the plants.The alreadyidentitiedspeciesirnd their principal host lcgumes are shown in Table I . The syrnbioticbacteriaseenrto havc e v o l v c dl o c a l l y ,c o i n c i d i n gw i t h t h c l e g u m e distribution. Thus. we cncounter native
Brudvrhi;tbiutn juytniturtt .lncl Rlti:obian lierlil (nodulatingsoybean)inChina.:"'Therc i s a l a r g c d i V e r s i t yo f R . e t l i l n o d u l a t i n g P h a s e o l u sb e a n )j n M e x i c o r r 'r 6 iL r ! l n d C o lombia.5l There are native populations of bv, viciae(nodulatingpeal R. LegLtninosarunr i n A i g h a n i s t a n r : : r 2arn do f R . g a l e g u ei n t h e Caucasus.rr5Wc may suppose that while Rltizobiun chromosomal lineages radiated and diversified,difl'erenttypes of symbiotic relationships may have evolved. In some cases,somebacteriallineagesbecamehighll' specializcd tbr sor.neplant hosts. A great deal o1' research has been devoted to R h i : o h i u m , B r a t i t r h i : . o b i u nl . a n d , 4 . : o r '
TABLE1 Bhizobium, Bradyrhizobium,and Azorhizobium Species" Species
Principaa l s s o c i a l e dh o s t l e g u m e s
R. meliloti Medicago,Melilotus, Trigonella R. fredii Glycine max and G. soja S. sahellb Acacia and Sesbania S. terangab Acacia and Sesbania B. leguminosarum bv. viciae Vicia and Pisum bv. trifolii Trifolium bv. phaseoli Phaseolus vulgaris R. tropici Phaseolusvulgaris and Leucaena R. etli Phaseolusvulgaris R. galegae Galega officinalis,G. orientalis R. loti' Lotusspp. R. huakuii. Astngalus snicus 8. japonicum Glycine max B. elkanii G. max A. caulinodans Sesbania rostrata " Other publishedspeciesare B. cicerilor Cicer arietinum and B. ttanshanensefot Glycne max. b A new generahasbeenproposedto encompassR. meliloti and R. fredii, and the new species S. teranga and S. saheli.Due to the taxonomicrules,the new generaname adopted must be the last one that was proposed and rejected,and correspondslo Sinorhizobium. " lt has been proposedthat R. /o and R. huakuiibe ass i g n e dt o a n o t h e rg e n e r a .
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hi:obiunt and severalreviews on their i n t e r a c t i o un i r h p l u n t sh a r e b e e n p u h -
introducedinto Brazil are nodulatedby the broadhost rangebacteriaR. trnpicirreand l i \ h e d . l i j . l a . a 6 .66lL. e l . l l r . l f ' 9 . 1 7 8 .a1S9 1W e l l a S spp. relatedto R. meliloti.sl by Rhiz.obium otherreviews concerning Rhi:obiunttaxNativeleucacnas. in contrast,arenodulated o n o m y l 1 6 . l l 8 1 2al n d e v o l u t i o n .l l ) sl .e 6 . : 2 8 which by strainsrelatedto R. alfi in Mexico,sr togetherwith other countriesof Central Americais the site of origin of Leucaena r7l B. Rhizobium Distribution species. It is sometimesdifficult to determine It is a puzzleas to whattheeffectsof whetherlhe Rhi:ohiunt p()pulrlion\ arenrpractices lgricultural havebeenon promot- tive to an areaor wereintroducedto it. This in{.Rlti:,'hittmpupulationsrnd il is lemptinP is the casewith R. tropiciTand with Rftl;oto speculatethat large densitiesof single biuntspp.relatedto R. elllrr7in Frenchsoils. species in cropshavebeena drivingforcein It hasbeenpointedout "how ignorantwe are selectingrhizobiawith narrowhost ranges aboutthemcchanisms by whichprokaryotes andhigherspccificities for somehostplants. aredistributedacrossthe Earthand the facThereis evidencethat man hascontributed torswhichinfluencetheirestablishment and to the spreadof rhizobiaaroundthe world. and difsuccess in a new envinrnment,"ree Supposedly, the Romanshelpedto spread ferentpossibilities for Rhizobiumspreadhave R. leguntino.sanun bv. viciae in Europeby been proposed.ree However, it still seems carryinethe soil as well as the pea plants reasonable to supposethat therehasbeena f r o m M i d d l e A s i a i n h i s t o r i c a lt i m e s . distributionof Rlizonaturalandsuccessful Plta.seolur vulgurisbeanwasintroduced lineagesindepenbium andBradyrhizpbium fiom Mexicoto EuropethroughSpainin the six dentof man. teenthcentury.rrIn Spain,thereare abunIn SouthAliica, R. troplci strains,iden(whereas tified by total protein profiles,have been dantnativepopulations ofR. e/lir5e no R. etli is tbund in Francerr0rrr recoveredfrom Bolusanthusand Spurtium nor in (still not recog"R. hairuntensis" Englandrro). It is known that B..jtLponicunt nodules.r8 il'asintroduced into the U.S.and llrazil as nizedas ir boru fide Rhizobiumspecies)r26 b a c t e r i alln o c u l a n t sw h e n s o y b e a nw a s has been isolatedfrom China,r0and it is adoptedas a maincrop in thosecountries.El clearlyrelatedto R. tropici by the l65 gene B. japonicum was also introducedinto andDNA-DNA homology.Origisequence Mexicoas a soybeaninoculantin the northnally,R. /r.?icl wasisolatedfromP. vuLgaris (commonbean)and from Leucaenalrees westernagriculturalareasapproximately 30 ycarsago, and in Poland,Italy, and India tiorn acid soils in South America.rreBoth withinthe last l0 or 15 years.loi'rr2 are\ntroduced P. vulgari.sandLeucaenatrees plantsrn theseregions,so it was proposed The introductionof cropsinto otherregionsmay havean impacton nativebactethat R. tropit:i u,as the natural symbiont of rial populations. hut the extentof theseef someotherlegume.R. lropl.l wasalsofound lectshasnot heencvaluated.ln somccases, in beannodulcstiom plantsgrown in acid the plantsnray recruit local "opportunistic soils in Kenya.raPossibly,the Portuguese introducedR. tropit'i to theseareasas well b a c t c r i a 't h a t h a v e c r o s s s p e c i f i c i t yt o plants. nodulatethesc This seemsto be the as the P. r,algarlsbeanplants.Also intrigucasewiththepeanuts introduced intoChinarnr ing is the fact that Agrohacterium isolates fiom tumorsof kiwi and cherryplantsfiom andwrth theLeucaenatreesintroducedinto dilterentregions.e5.|6r The Leru:aenaplants as well asAgntbacteriuml:it'tvar 2 Japan,lTr
' |1 5
strains, are among the closest relatives to R. topi(i strains.as determined by thc seq u e n c eo l l 6 5 r i b o s o m a lg e n e s . r r r : rO r ther similarities among these agrobacteriaand R. tropit'i have bccn lbund as rvell.rrsThc geneticdif'terencesthat determinewhethcra bacteriumis a pathogcn or a svmbiont arc discussedlater. There are more examplesof a wide geographic distribution of other Rhirobiuntlit'teages.The sametypes ol R. leguntirtosorum have been encounteredboth in the U.S. and in the U.K.reeSorre isolatestrom Metlir:ago ruthenicufrontMongolia are relatedto R. zrll (bv sequenceof l65 ribosontal gencs) ancl the majority of M. rutlpnita isolatesare a lineaqebctween R. rlli and R. tropir:i.:t\iht Sor.rthAfiica, R. elll (identificd by protein profiles)hasbeenisolatedfiom De.vnotliunt, Memolohiunt, and Indigo.fera and fiom the t r e e f e g u m e s A c o c i u n e l . u t t o . r t . 1 o na n d Clrumuecristn.strictu)Elt would be interestine to analyzethe South African isolatest0 dctermine if they are similar to the Mcsoamencan R. elll isolates in othcr respects. R. etli like symbiontsalso have been identified in Argentina liom bean nodules.rOriginally,R. erli was found nodulatingP. r,ulguris plantsin Mesoamerica.rs0 Bacteriarelatedto fi. arli have been isolatedfiom difl'erentnative Mesoamericanlegumest,'and from bean nodulesin France,llTThese bacteriaare diverging lineages that, we suppose. had a common ancestorwith R. etl/. As mentionedpreviously. B. japonitunt diversifled in Asia. Therc has been a succ e \ s l u l B .j . / p , a l t ( , , i n u c u l J l i u n r c \ p o n s c in soybeancropsin otherregionsofthe world. The responseto inoculationhasbeen linked. in the majority ofthe cases,to the absenceof native bacteria with the ability ro nodulate the introducedcrop.8rln Mcxico, there are no natlvepopulationsofB. japonlo,ri;howcver, native R rady"rhizobiutnhave been rccovered liom native Lirp lris plants. These bacteriaare closely related to B. jqtonicunt, and we estimate that they have bccn geo-
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g r a p h i c a l l yi s o l a t e clli o n r B . j u p o t t i t u m f < : r m a n y m i l l i o n s o f v e a r s .Y c t . t h e ) ' d i f l e r b 1 ' o n l y o n e n u c l e o t i d ei n a i i a g m e n to l t h e l 6 5 r i b o s o m a lg e n c s( o u r o r v n u n p u b l i s h e dr e s u l t s ) .L i r p l r r r . ii s o l a t e sf r o n r o t h e r r e c i o n s are clearlv related to Ilrulyrhi:oltiurrt as s'ell. oo
C. RhizobiumGeneticDiversity The wide geographrcaldistribution ol' Rlti:obiuntirnJB rurlrrhiTobiutnbacterian.]ay b ( rr e l a t e dt o t h c l a r g c s e n e t i cd i v e h i t v e n c o u n t e r e di n r h i z o b i a .w h i c h r s a c o l l n t o l l l r n d i n g r v h e n a n a l y z i n , et h e s e p o p u l i r l i o n \ r 6 l rl i r i r L i] ) s . r i ' ! . r(,s.cecs e c t i o nI I 1 .T h e i r l a r g e b i o d i v e r s i t y .a s * e l J a s t h e i r b r o t d g c o g r a p h i c adl i s t r i b u t i o nr r a v b e i n t e r p l c t c d as evidence th,trt Rlti;.obiunt and Brrllr'rhi:ttbiunt are ancicnt lineages ol bacteria w i t h a l o n g c v o l u t i o n a r yh i s t o r 1 , . r r " A singlc lcgumc. for cxample. ..lclcla, s o y b e a n o, r L e u c u e t t u c. a n p i c k u p l r e n e t i c a l l y d i s s i m i l a rs v m b i o n l . sT. h e r e l b r e . ' , v i t h a terv legume species.we have sanrplesof l a r g e b a c t e r i a l h e t e r o g e n e j t v .A c a c i r r i s n o d u l a t e db y B r u d t r l t i . o b i r a rs p p . , r r i 5 1br v: r R h i z o h i u n r e l a t e dt o R . / o t l ( c l u s t e rU ) . r l by Rhi:obiunt related to R. meliloti lthc r e c e n t l y p r o p o s e d S i n o r l t i : . o b i u r t ;t u h e l i ^nd lerongut:\. and by R. /lrialali. Soybean i s n o d u l a t e db y w i d c l _ vd' i v e r g e n tb a c t e r i a , B - . j n p o n i t ' t r n t , tB t t.ae l k u n i i , t ) ^" 8 . l i u o t t i r t t "[]. titutshunense'tt (the last trvo ,gtr.re,"r n o t y e t r c c o g n i z t da s b o n t Ll i d e s p e c i e s ) , r r " lnd lsy R. lretlii.')t')\ttLeLu:tLettL is nodulated b y a w i d e r a n g e o f r h i z o b i a . r L\ - . ' . ri r5e ' r r l T h e d i v e r s i t l ' o f R h i . . o h i u nn t oclulating /'. r,ulgrrn,r' hasnot bccnconrpletell'clcscribcd a n d , i n a d d i t i o n t c t R . t r o p i t i , r t ' )R . r , / i l .s ( r i\nd R. legatlitlo.ralrinlbr,. phascoli.rl)rtherc a r e n c w f i n d i n g s o f l i n e a g e sn o d u l a t i n g b c a n . 5rlr . r ) r , H i g h g e n e t i cd i v e r s i t yi s n o to n l y a p r e l a , lcnt charactcristicof tropical legunre symb i o n t s .b u t i t i s a l s o a c h a r a c t c r i s t ioc1 ' s y n t -
l . r i o n t sl i o n t t c t n p e r a t el e g u m e s . : r s u c h z r s . 4 s t u q u l t rs s p p . . O r t t l r 4 ; l . r t u r n p a r t u l a t u . H , r l t r , t r t l t t , t l l , t r t t t n t (- , l r r l l i r , i l r t r r r l i . Clycyrrlri:u spp.. Coronillu l (/rld. and 1 . r r 1 r i r l .si p e c i e s R . . htnkttii was idcntified 1nrrn,4.r'trrr,ga[r.i .r'lrriaisfrom Chi na]')and it (from Lorrr.ris relatedto R. /r.,1r'rr spp.) by the s e q u e n coef l i b o s o m t l g e n e s M . orerecently, R . r ' l cr r r . ) 5 5r e l a t e dt o t h i s c l u s t e rb u t n o t v e t o l f i c i a l l v r e c o g n i z c da s a s p e c i e s . r 1wL a s plol.rosecl to encornpasspart of the symbtonts o b t a i n e df r o r n C l r z r r t r i t t i r t u n t( c h i c k p e a ) . r\s tttore strains from A;trugulus have bccn r . ' n s i J c t ( dl i r r l h e i rI i b o ; ' l t t t asl c q u e n c e i.t. has Lreenfbund thatA r/r.t,qllls and chickpea isolatesare not really separatecl. but that they o v e r l a p . r ' )A r ' r e c c n t l y d c s c r i b e dg r o u p o f r h i z o b i a( " R . t i u n t h t t t t t ' t r . t t i ' t tn) o. t y e t r e c o r n i z e da sa s p e c i e s . r r i ' o r i g i n a t iinnga n a r i d rnd suline environnrcnt in northwestern China. rvaslirund to be relatedto the R.1r.,liR . l t r r t t k t t ici l u s t e r .l t r e m a i n s k ) b e e s t a b l i s h c d i f t h e O i r o r r i ,iis o l t t e s .f e l a t e dt o t h c R.lt,r,tl,trtR ' .,i{ { /t LIu\l(t.
'
er)n\lilllle il
d i s t i n c t b r a n c h .B y o t h c r c l i t c r i a . s u c h a s p h a g c t y p i n g . R . / o r l i s r e l a t e dt o s e v e r a l A.st ruga I u.s.H cd t,su rum, and (Jnr.,nl.r rhizitb i a a s w c l l u s t o R . h u u k u i i s t r a i n s . r fF r 'u r thcrmorc.R. /()11 strainsare able to nodulatc O . r r r l t , n s r . i . r i rA' . st r u g a l t ts . I l e d l : ; t r u n t , ( i l y t y r r l i . u . a t l d O n o n i . \p l a n t ss e e n rt o b c I o n g t o n s i n g l ec l o s s - n o d u l a t i ognr o u p . A n o t h c rc r a r l p l e o l ' v a r i o u sd i s t i n c th o s t l c g u n r c sn o d u l a t e db ) a c l L l s t e ro l r e l a t e d bacterir is the caseol the Mesoamcrican l ( . - r r l I r e/ \) , / / ( i /l , 1 ' , ' v, , , , -C l i t , ' r i , tI t r t n t l L rt( rotult riu spp..Mucrollilitrnt uil:tbosiloliutn. i\nd Leu(u(no spp.. whose synrbiontsare a g r - o n po l g e n e t i c a l l vi n t e r m i n g l e dr h i z o b i a c l o s e l vr e l a t c dt o R . e t l i . f r ' H e t c r r r g c n e i l vi n s i d e B . j r t l t o n i c t u t t s e r v e da s a b a s l st o p r o p o s eB . e l k u n i i . i t : t 1 ^ B xttly rlti.ohitutt *'ith specificitl, fL.,rVi.Ettt. l ' . I u n t tI t r . s . .r1r r r ' l i i . ra,n d M u t n t p ti I i ut n l r r e , t h i g h l y h e t c r o g e n e o ugsr o l L po l b a c t e r i i l . r l r l P h o t o s y n t h e t irch i z o b i al l e i n c l u d e du i t h i n t h c B r u t l v r l t i : o h i l l / c? lru s l e r .S o n r eo l t h e n r
perhaps an independent branch.L'0 constitute A large numbcrof native legumesfrom by 8,7ttlvrhi:.obiunt Anrazonia arenodulated strainsthat do not correspondto the describcdRraA rftl;oblunl-proups.ir5 Basedon sequences of l6S rRNA genes. thecomplcte a distinctbranch(f Brutlvrli:obiwnhasbeen includingB. elkanrrand isodistinguished, latesfionr Astrugalusand from a Manchurian tree.lo('
D. Grouping Rhizobium At prcscnt. Rhi:.obiuntand Brutlyrhi:obiun lineageslook quite complicated. yetonlv a srriallpercentagc of thenodulating lcgumcshavebeensampledtbr theirrhizobia.Furthermore, it hasbecnclaimedthatby lookingonly al theisolatestiom noduleswe may be overlookingdiversity,as perhaps only a snrallproportionof the wholepopul r l l o n .r h cr ) n cu i l h t h eh i g h e ret a p a e i tl lo nodulate,is analyzed.Actually, this is the casclit hasbeenshownthat the R. meliloli populationobtainedfrom plant nodulesis not representative of the total population r'r prcscntin soil. We mustexpectthat in the ncarfuturc,we will haveto deal with an diversecollection of rhizoovcrwhelmingly h r l . I l . c , . r n lrh r t r n i r n \, r f t h en e u s p e c i c s rvill beisolatedliom thetropicsandsubtropi cs .l r ' l M i c r o b i a l b i o d r v e r s i t yh a sb e e nc o n s i d e r e d a s o n e o l - t h e m o s t v a l u a b l er e s o u r c e s f b r m a n k i n d .Y e t . l i r r p e o p l c w o r k i n g w i t h Rlti:obiunt LrndR rudy rh iaobii.rrr.tt ts very d r t T i c u ltto h a n d l es u c hd i v c r s i t y ,a n dp r o p e r a n d s i m p l e r m e t h o d st o r d e n t i l y a n d c l a s sify Rhi:.obitunbacteria need to be develo p e d . A t p r c s c n t . r e s e a r c h e r sh a v e m o r e t o o l s a n d c r i t e r i a t o a c c o m m o d a t eR h l : o /)irril slrains rn biologically meaningfui g r ' o u p \ . 1r6l r r e N L :r r 1 y) e t t h e g e n e r a l p r i n ciple is the same that was used about a ( ( n t u r ) i r s o l , r r l c l i n en h \ l ( ) g e n i c 3\ m o n P
'117
J i v i n go r g a n i s m s . s r . rt rh6e n t o r ec h a r a c t e r i s _ c o m p r l s r n tll6 g e n c sr v e r en t a p p e di n s i n r i l a r t i c s t h e y s h a r e ,t h c m o r e s i n t i l a r t h e v a r c . p o s j t i o n si n t h e t c s t c ds t r a i n s . rrlr r r r ) r, ) tU n N , , r r ' , f . i r c r c e J t h i l t R / r r . r , / , , r i rlri r r . . a g e . 1 i ; r t u n a t e J l t. h c s e e x p e r i r n e n r sw e r e p e r _ s h o u l db e e s t a b i i s h e b d a s e coi n s r a b l c( c h r o tornrcciwhen onlv I lintitednurnbero1Rll.:r.,_ mosomal)properties.rir.\ Thc prescnttrcjtds ,1r{/l speciesrrerc known. It rl,ouirjbc a r e c o n s i d e r i n gm o r e g e n e t i ct r a i t si n a d d i _ i n t c r c s t i n gt o h a v e t h i s t y p e o f i n f b r m a t i o n tion to phenotypiccharacteristics. but in sev_ ( g e n cm a p s )l r o n t t h e o t h e rn e w s p e c i e sa. n d crai cases. phcnotl,pic_and genctic-based t h e s cl v o u l d b e s t i l l n t o r e v a l u a i r l ci f r l o r c groupingsdo not seemtclmatch.rr,rr./i).1r0 r.i\gcnetic markcrs rverc considered.as well as ing controversyas to the uscl.ulnessof tlrc t h c l i e c l u e n c i cosf c h r o m o s o m arle c o m b i n a p o l y p h a s i ca p p r o a c ht o d e f i n c r h i z o b t a spc_ t i o n a m o n g d t l ' i c r e n ts p e c i e s I. t w o u l d a i s o ilr'll(l cie\. I ' r ' u s c l i t tl r ' l r r r ' u i l c h r r , r t t , , , , , r r t c , i /acr,r , From thc presentkno*,leclgeof bacterial e q u i v a l e n ti n d r i f e r e n tl i n c a g e sa s a n a d d i _ genctrcs.we predict tltat even a tew genetic tional support tirt1.. 1 5 . 1 2I.9 9 , 1 .
2 3 . Carlson.C. R., Caugant,D. A., and Kolsto. A.-l]., Ccnolypic dtr crsitl among Bocillti.r r cn us itttdIlacillu.stru,t,qi('/r.\ir straios.,'1121. [int intt. lt4itroltiol.,60. l?19. 1994.
tl
Carlson, R. W., Price, N. P. J., and S t a c e y .G . , T h c b i o s l n t h e s i so l r h i z o b i a l lipo oligosaccharidn e o du l a ll o r l s l g n a I m o l e c u l c sM . ol. Plant Mitrobt lnleratt-. 7 . 6 1 J 4 l.9 g . l .
129
2 5 . Caugant, D. A.. Bovre. K., Gaustad, P.. Bryn, K., Holten, E.. Hoiby, E. 4.. and Froholnr,1,.C),,!lultilocrrsgenotlpe\deter minedby enzyne electrophoresis of Ncirst,riri tnettitrqitidis isolatcdilonr pilticnts\rith \\s temic diseascand lronr lrcallhl carricls../ . 986. G e n .M i o o b i o L . .l - l l . 6 - 1 1 1
26. Caugant,D. A,, l-roholm, L. O., Bovre,K., IIolten. E., Frasch, Cl. E., Nlocca. L. F.. Zollingcr. W. D., and Selander.R. K., In tercontioental spreadof u genelicrillydistincl i v c c o m p l c xo l c l o n c so l N e i s s e r inat e n r n .grlilij cirusingepidcnricdiscasc.Prot. Notl. rlrrrl. .lti. U.S.A..8-1.'1927.1986.
2 7 Caugant,D. A.. Levin, B. R., and Selander,
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latcsirl Mlrrrbrtttr'r'itrntleltrat'..1Iltt tarirtl.. l l I . 1 t , 1 . 11. 9 8 9
-t+
Porrs,.l.-L..Sesboue.Il.. and N l a r t i n , . 1 . - P . ,I i l e c t r o p h o t e t i ct r r n \ l e f I r o n r p o l t a c r l - l a n r i d eg c l l o n i t r o c c l l u l o s es h c e t s i.r r t e * m c t h o c l l o c h a r a c l c t t z er l l u l { l l ( J c L el \n za'me genottpe\ ol Klalt..it'llu stttin:. A2|/ l',nt innt. l\licntltto[. (r(]. 16. 199-1.
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1., Diflerentiation bctr,"cen A ltirrr.rbalillrr.i ( H a e n t o p l t i I u . st I( t i n ( ) 1 \ ' a e t c n t a| ) n it I L t . t , Haernoplilus qtnrythilus and Haet q)hilu.l pttrupltntphilusb1 multilocuserlzymeelect r o p h o r c s i sJ . G e t t .M i t n t b i o l . , l l 6 . 2 1 3 5 . 1990. : 9 . Chen, !V. X., Li, G. S., Qi. Y. L., Wang, E . T . , Y u a n ,H . L . , a n d L i , J . L . . R h i : o l t i u t t t /rrro&rrii sp. nor'. isollted from the rool nod ulcs ol Astrrrgzrftr.isirricrrs./rrt. J. SrJ/. Bacreriol...l I . 275. 199L
3 0 . Chen, W. X., Wang, E. T., Li, Y. ll., Li, Y., and Gao. J. L.. Rhi:.obiunhauutnensis. A new speciesof root nodulebacteriaisolated from tropicalregion.in Pnr'. Int. $n4t. Di tersitt uul T utonontyr,,/R/ri:obia.Wuhan. H u b c i .C h i n a .I 9 9 - 1 .1 9 .
3 1 . Chen,W., Wang, E., Wang,S.,Li, Y., Chen, X,, and Li, Y.. Chirracteristics ol Rhittbinn /i.'i.sll.rllr.r? sp.nov.. a rrxrderatelyandslorvlygrowingroot nodulebacieriumisolatedfronr a n l r i d s a l i n e e n v j r o n o r e n ti n X i n . j i a n g . P c o p ) c ' sR e p u b l i co f C h i n a . 1 r l . . / . S r . ! / . B o c t e r k t l . . . l 51. 5 3 .1 9 9 5 .
1 2 . Chen, W. X., Yan, G. H., and Li, J. L.. N u n r e r i c rt .l r \ r l , , n l t \ 'l u J \ , ' l l . r . ti t , ' u i l l ! soybeanrhizobiaand a proposalthat Rliao bium lredii be assignedl,,J Sinorhi.t)hiLlnt gen. nov.. /rr1../. .S1\1.Ilttrteriol.. 18. 192. l 98 l J .
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E., ll,lourson. .{. .I.. and'fhompson, J, K.. Idenlilicirliort ol /,/)ltr.rt hizohilt br dilcct DNA h,''bridization of cfLrshcd rrxrt n o d u l e s ,. 1 2 1 ; /I.' , r t r i n n t l.i i L u t l i o l . i l . I 7 0 5 .
R, K., Gcneticdirersity ancltemporallaria tion in the f cr.,/ipopulationofr humanhost. C c r e l r rs . 9 8 . 4 6 7 . I 9 8L
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C0han, F. \1.. (;!'l]allc c\cl]lill!a ilnd c\olLl_ tionlr) di\ergcncc in lfoklf\ olc\. 7)('/r./.r I t o l L t t l . 9 . 1 7 5 .1 9 9 . 1 .
t987. l 7 Crorv,V. l,.,.larris, Il. D. w.. and (;reen' acidhonrolo rvood,R. N1.,Deoxltibonuclcic gies among lcid producitrg \tfarn\ ol lilli:/)' l t i u t n . l n t . . l .S l s t B u t t t r r o l . . 1 1 . 1 5 1 . l t ) E l .
38
Dagutat, H. and Steyn, ['.L.. lltronortlt i , L n dd i s t r i b u t i o n o t r h i z o b i r i r r d i g e n o u sl o S o u t h A l r i c a r t s o i l s . i n , V i l r o g e t tl l t r t l l / r t t : -[ atrrrr.r. tkhonor tch. l'undonxttltIs und A7.,;.,/ir L A. cr rl.. Ilds.. Kluucr. The Nctherlands. 1995.681.
1 9 . D a k o r a . F - .D . P l r n t l h r o n o i c l s :b i o l o g i r l l n l o l c c u l e sf b l L r s c l ucl x p l o i t a t i o n . , ' l r t ..r/t.. P l a r t tP l t v . t i o l2. .2 . 8 7 . 1 9 9 5 . .1t.1.D c b e l l 6 , [ . , R o s e n b e r g . C ] . , V a s s e , . l . . N{aillet, F., \lartinez. E., Denari6,.J.,and of sl mbioticcicrc'l Truchet, (i.. Assignrncnt phcrtotl,pes lo conrnrrn itnd spc opmental (/r,./) cific nodulation Scnclicloci ol R/ri.-rr biun nteliItti. J. IJed(t'i()[. l(r8. l(]75. l9ti6 D e t t r u i j n .F , . 1 .|. . e . r l t t r ' , . t t t r tr ,r e n c t r l r \ ( ' extragcnicpalindromic aId cllleroblrcterirl repetitivcintergencriccon\cn\Lr\)sequcnc!'s lnd the poll'mcrasechlin fcrction 10iingef' print thc llenomcsol RlttltbiunrDt.liloli 1\o l .t t v i n n t . l i r t c sa n d o t h c rs o i l b u c t e r i aA. 1 t 1 tE M t c n t l n t l . . 5 8 .I l 8 { ) , 1 9 9 1 . .11 D c l , a j u d i e . P . , W i l l c m s , 4 . , P o t , l t . , l)e$ettinck, D., Nlaestrojuan,G., Nelra, N{., Collins, NI. D., Dreifus. B., Ktrsters. K . . a n d ( l i l l i s , l \ 1 . .P o l ) p h a \ i cl a \ o n o n r \o i
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f hrzobia:ernendirtiou ol the -qenus .tt,lor-lli:obirar anddescliption o[ Sinrtrli:obiummelikni conrb. no\.. Sinorlti;.obium salrell sp. nov.. ind Sinorhi:obiun terdnqu sp. nov.. 1rL J. St.sr.BLtL rt'riol..:1.1. 7 15. I 994. 'f.?-r.)r, -1-l De l,ey,.J..Phylogenyol prokariotes, 2 3 . 2 9 1 .1 9 7 : 1 . Dcmezas,D. H., Reardon, T. 8., Strain, S. R., Watson, J. M., and Gibson, A. H.. D i \ e r s i 1 )a' n d g c n e t i cs l r u c t u f eo f a n a t u r a l populrtionof Rhi;obiuntIeguntitrosarun bt. r/ toli I isolatedfronTrifoIiunt subterraneurn L . . M o l . E c o l . . . 12. 0 9 . 1 9 9 5 . .15. I)emezas,D. H., Reardon,'I'.8., Watson, J. NI., and Gibson, A. H., Gcnericdiversity anone Rhi:ttbiwn Legumi |)sarun br'. trifolii strarnsfelealed by allozynteand restriction trrgn]cntIengthpolymotphisrn anaJvscs. Ap1. l',ntirort.Mi
