Numerical Taxonomy of Rhizobium Strains from Legumes of the Temperate Zone

Transcription

1 NTRNATONAL JOURNAL OF SYSTMATC BACTROLOGY, Oct., p. //$. Copyrght, nternatonal Unon of Mcrobologcal Socetes Vol., No. Numercal Taxonomy of Rhzobum Strans from Legumes of the Temperate Zone NATAL. NOVKOVA," LNA A. PAVLOVA, NKOLA. VOROBJV, AND LNA V. LMSHCHNKO Research nsttute for Agn'cultural Mcrobology, St. Petersbug, Russa Phenotypc propertes (growth characterstcs, utlzaton of carbon an ntrogen sources, an ntrnsc antbotc resstance) of Rhzobum strans solate from root noules of the temperatezone legumes Astragalus spp. ( strans), Oxytrops campanuhta ( strans), Heysarum alpnum ( strans), Onons awenss ( strans), Glycyrrhza spp. ( strans), an Coronlh vara ( strans) were compare wth those of other Rhzobum, Brayrhzobum, an Agrobacterum strans. The fnal matrx, contanng strans an nonsymbotc features, was use for computer cluster analyss. The enrogram showe that the new strans of temperatezone rhzoba forme a cluster separate from both Rhzobum an Brayrhzobum spp. Two large groups of temperatezone rhzoba were reveale. Group nclue rhzobal strans orgnatng from fferent geographcal regons wth a temperate clmate, whle group nclue strans from the same geographcal orgn, South Sbera. The 'strans of recognze Rhzobum speces were clustere, n general, wth each other as expecte from phylogenetc relateness. Root noule bactera of the genera Rhzobum an Brayrhzobum are a common mcroflora of temperate sols. These bactera may nteract wth the roots of legumnous plants, nucng the formaton of noules n whch the fxaton of atmospherc ntrogen occurs. These bactera are commonly use n agrculture for mprovement of plant growth. The specfcty of the nteracton between rhzoba an legumes epens on many factors coe for by the host cultvar an bacteral genomes;. The man propertes of a bacteral solate can be escrbe by ts taxonomcal poston n relaton to other strans. There are several Rhzobum speces recognze at ths tme (,., ), an knowlege of rhzobal evoluton an phylogeny obtane n recent stues allows the proposal of new rhzobal taxa (,, ). However, the current systematc escrpton of the genus Rhzobum oes not nclue many of the root noule solates from legumes of tropcal (, ), arctc (), an temperate (,, ) regons. The host plant specfctes an phage senstvtes of Rhzobum strans from temperatezone legume root noules forme on Astragalus, Heysamm, Glycywhza, an Onons plants were recently escrbe (). t was shown that these strans consttute a crossnoculaton group relate to Rhzobum lot. The am of the present work was to etermne the cultural propertes of these strans an to compare these propertes wth those of other Rhzobum speces. MATRALS AND MTHODS Bacteral strans. Bacteral strans use n ths work are lste n Table. Bactera were mantane on yeast extractmanntol (YM) () agar slants at C. Growth characterstcs. The tme necessary for full colony evelopment on YM agar (estmate here as a colony ameter of mm) was etermne. Bacteral growth on meatpeptone agar an reactons wth ltmus mlk were etermne as escrbe earler (). The ablty of strans to grow * Corresponng author. Present aress: Centro e nvestgacon sobre Fjacon e Ntrogeno, UNAM, Apo. Postal A, Cuernavaca, Morelos, Mexco. Fax: (). lectronc mal aress: novkova@n.cfn.unam.mx. n the presence of,,., or % NaCl was teste by streakng strans on YM agar contanng the salt. Temperature tolerance was etermne by platng strans to obtan sngle colones on YM agar an ncubatng at,, an C for to ays. Unless ncate othenvse, the cultures were ncubate at C. Growth of the fastgrowng an slowly growng strans was observe after ncubaton for to an to ays, respectvely. All tests were performe n uplcate. Utlzaton of carbon sources. For analyss of carbon source utlzaton, the base meum (YM) was mofe so that yeast extract was replace by.% NH,NO, an manntol was replace by one of the followng carbohyrates: sucrose, Dmaltose, Dlactose, Dglucose, Darabnose, Drhamnose, D mannose, Dxylose, Draffnose, ulctol, Dsorbtol, or the carboxylc ac Nasuccnate. The mea were solfe wth.% agar, an.% (wt/vol) bromothymol blue was ae as a ph ncator. The ph of the mea was ajuste to.. Tests were one n tubes. Utlzaton of amno acs as sole ntrogen an carbon sources. Tests were performe wth the same meum use for the etermnaton of carbon source utlzaton, but the ntrogen source was replace by one of the followng amno acs (.%, wt/vol) as a source of both ntrogen an carbon: Lhstne, DLvalne, Largnne, Lprolne, or DLserne. Utlzaton of mneral ntrogen. The yeast extract n YM agar was replace by.% (wtlvol) of one of the followng mneral salts: (NH,),SO,, Ca(NO,),, (NH,),PO,, or (NH) MOO,. The mea contane.% bromothymol blue as a ph ncator. ntrnsc antbotc resstance. ntrnsc antbotc resstance was teste on YM agar supplemente wth one of the followng antbotcs at the ncate concentratons (n mcrograms per mlllter): streptomycn,,, or ; rfampn,,, or ; kanamycn,,, or ; penclln, or ; or ampclln, or. Noulaton tests. Legume sees were obtane from the Vavlov Plant Breeng nsttute (St. Petersburg, Russa). Tests were one n tubes wth surfacesterlze legume sees as escrbe earler (). Twelve seelngs of each legume were noculate for each stran teste. Tubes were examne for the presence or absence of noules after weeks. The ablty to fx ntrogen was estmate by measurng acetylenereucng actv Downloae from by P:... On: Tue, Nov ::

2 VOL., NUMRCAL TAXONOMY OF TMPRATZON RHZOBUM STRANS Brayrhzoburn japoncum strans CAM CAM Brayhzobum sp. strans (Desmoum) CAM CAM CAM TABL. Rhzobal strans use n ths work Strana Host plant Geographcal orgn an/or Cluster reference Rhzobum tropc CAT = Agrobacterum tumefacens bovar C Rhzobum J;e USDA Rhzobum sp. stran NGR Rhzobum huaku CCBAU Rhzobum galegae strans HAMB T CAM Agrobacterum raobacter bovar K Agrobacterum rhzogenes bovar ATCC Rhzobum mellot strans CAM CAM su Rhzobum fre Rhzobum legumnosamm bovar vcae CB Rhzobum lot strans NZP = NZP Rhzobum huaku strans CCBAU CCBAU T Rhzobum sp. stran CAM Rhzobum legumnosarum bovar trfol strans CAM CAM Rhzobum legumnosamm bovar phaseol strans CAM CAM Rhzobum sp. strans CAM CAM CAM CAM CAM NZP (=CC ) CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM CAM Glycne m u (L.) Merr. Glycne m a (L.) Merr. Desmourn canaense (L.) DC Desmourn canaense (L.) DC Desmourn canaense (L.) DC Phaseolus vulgars L. Glycne max (L.) Merr. Lablab purpureus Astragalus sncus L. Galega orentals Lam. Galega orentals Lam. Mecago satva L. Mecago satva L. Mecago satva L. Glycne sp. Psum satvum L. Lotus tenus Kt. Lotus varcatus Astragalus sncus L. Astragalus sncus L. Heysamm alpnum L. Trfolum pratense L. Trfolum hybrum L. Phaseolus vulgars L. Phaseolus vulgars L. Astragalus ulgnosus L. Astragalus falcatus Lam. Astragalus ulgnosus L. Lotus comculatus L. Anthylls arenara (Rupr.) Juz. Coronlla vara L. Onons arvenss L. Glycyrrhza glabra L. Astragalus poltov Krl. Astragalus fngus Bge. Astragalus ulgnosus L. Heysamm alpnum L. Heysamm alpnum L. Onons awenss L. Onons awenss L. Glycyrrhza pallflora Maxm. Glycyrrhza pallfora Maxm. Glycyrrhza pallflora Maxm. Coronlla vara L. Heysamm alpnum L. Heysamm alpnum L. Heysamm alpnum L. Heysamm alpnum L. Coronlla vara L. Lotus comculatus L. Downloae from by P:... On: Tue, Nov :: Georga () Georga () Ukrane (ths work) Ukrane (ths work) Ukrane (ths work) Colomba () Chna () New Gunea () Chna () Fnlan () stona () Kazakhstan () Latva () Australa () na (ths work) nglan () New Zealan () New Zealan () Chna () Chna () Moscow, Russa () St. Petersburg, Russa () Byelorus () Armena () Armena () Vorkuta, Russa () Stavropol, Russa () Alta, Russa (ths work) Moscow () St. Petersburg () Unte States () Lpetsk, Russa () Turkmenstan (ths work) Novosbrsk, Russa () Byelorus () Byelorus () Byelorus () Vorkuta () Ukrane () Ukrane () Ukrane () Moscow () Moscow () Lpetsk () Lpetsk () Ukrane () Ukrane () Ukrane () Daghestan (ths work) Moscow () Moscow () Moscow () Moscow () Daghestan (ths work) St. Petersburg () b Contnue on followng page

3 NOVKOVA T AL. NT. J. SYST. BACTROL. TABL Contnue Stran" Host plant Geographcal orgn an/or reference Cluster A A A A A A CAM CAM A A CAM CAM CAM CAM CAM CAM A A A A Astragalus ulgnosus L. Astragalus ancus Hetz. Astragalus austrosbrcus B. Oxytrops campanulata Vass. Oxytrops campanulata Vass. Oxytrops campanulata Vass. Oxytrops campanulata Vass. Oxytrops campanulata Vass. Oxytrops campanulata Vass. Astragalus ulgnosus L. Astragalus ancus Hetz. Astragalus ancus Hetz. Astragalus ulgnosus L. Oxytrops campanulata Vass. Astragalus falcatus Lam. Astragalus ancus Hetz. Astragalus falcatus Lam. Astragalus falcatus Lam. Astragalus ancus Hetz. Astragalus ancus Hetz. Astragalus ancus Hetz. ~ ~~~~~~~~~~~~~ ' CAM, stran obtalne from the culture collecton of the Research nsttute for Agrcultural Mcrobology, St. Petersburg, Russa. Strans of R. huaku were receve from.t. Wang (Bejng Agrcultural Unversty, Bejng, Chna). Other Rhzobum strans were prove by K. Lnstrom (Unversty of Helsnk, Helsnk, Fnlan). T, type strans of speces. ", Stran not part of a cluster. ty. n all plant tests, unnoculate plants were nclue as controls. Numercal analyss. A computer cluster analyss of phenotypc nonsymbotc varables for strans was performe by usng the weghe completelnkage metho as escrbe earler (, ). The ata were coe for bacteral growth characterstcs as follows: for an absence of growth, for lght growth, an for heavy growth. Changes n the ph of the culture mea were assgne values of for strong acfcaton, for a weak acfcaton, for no change, for weak alkalnzaton, an for strong alkalnzaton. Reactons on ltmus mlk were gven values of for the absence of a serum zone an for the presence of a serum zone. Growth rate values were assgne on the bass of the number of ays t took for full colony evelopment to occur. Weghtng coeffcents use were for acty of culture mea, for growth rates, an for other characters. The characters wth a sperson of were exclue from the calculatons. The analyss was performe at the Computer Laboratory, Research nsttute for Agrcultural Mcrobology, St. Petersburg, Russa. RSULTS Computer analyss allowe us to group the strans nto clusters on the bass of ther metabolc propertes. The composton of each cluster s shown n Table. A enrogram showng the lnkage between clusters s presente n Fg.. The strans uner nvestgaton forme two large taxonomc groups wth a. level of smlarty. These groups were foun to be vergent both from other Rhzobum speces an from Brayrhzobum strans. The level of smlarty between these groups an the Rhzobum reference strans was about., an the level of smlarty between Brayrhzobum an Rhzobum speces was less than.. The metabolc propertes of the strans are presente n Table. Cluster nclue slowly growng Brayrhzobum strans solate from root noules of Desmoum canaense an Glycne max. Ths cluster has the greatest vergence from the other rhzoba teste. Cluster consste of only two strans: Rhzobum tropc CAT an Agrobacterum tumefacens C. Cluster comprse strans havng fferent taxonomcal postons: Rhzobum fre USDA, Rhzobum huaku CCBAU, an Rhzobum sp. stran NGR. Cluster nclue four strans: two strans of Agrobacterum an two strans of Rhzobum galegae. Cluster s compose of Rhzobum mellot as well as R. fre strans. t shoul be note that strans from fferent geographcal orgns but belongng to the same taxon were, as a rule, clustere wth one another. Cluster comprse the relate strans of R. lot an R. huaku. Clusters through unte nto one taxonomc group wth a level of smlarty of about.. Ths group nclue nearly all of the Rhzobum an Agrobacterum reference strans use n ths work. Two Rhzobum strans (CAM an CAM ) were not clustere wth one another or wth other bactera teste an occupe stnct postons on the enrogram. Cluster nclue two Rhzobum strans solate from root noules of Phaseolus vulgars, two strans entfe as Rhzobum legumnosarum bv. trfol, an two solates from root noules forme on an Astragalus sp. Ths cluster was more relate to the Rhzobum sp. strans uner nvestgaton than to the reference strans. Downloae from by P:... On: Tue, Nov ::

4 ~ VOL., NUMRCAL TAXONOMY OF TMPRATZON MZOBUM STRANS Level of smlarty Clusters,, j, j!! j!!,.,. :. j ClAM A A A OAM s A CAM AL ClAM A OlAM OlAM ClAM A Af All A A ClAM A OAM GlAM OAM OlAM l. OlAM CAM aam OZO ClAM ClAM OlAM ClAM OAM s ClAM ClAM OAM ClAM. OlAM L. OAM OAM OAM OAM L OlAM L NZP S OAM L ClAM ClAM OlAM ClAM lol a Group Group a WAM tl UAM a WAM OAM OlAM OAM. R.legumnosarum AU CCBAU NZP Ce SQ NZP su OAM ClAM OAM HAMB USA NCR M OOBAU USOA OlAT Rhzobum Agrobacterum Bra yr h zobum FG.. Denrogram reflectng phenotypc smlartes between Rhzobum strans from temperatezone legumes an other Rhzobum, Agrobacterum, an Brayrhzobum strans, Downloae from by P:... On: Tue, Nov ::

5 NOVKOVA T AL. NT. J. SYST. BACTROL. Characterstc TABL. Metabolc propertes of clustere strans" Downloae from by P:... On: Tue, Nov :: Cluster (n) ~~~ ~~~ ~~ l() () () () () () () () () () () () () () Acfcaton of meum contanng the followng carbon sources: D Arabonose DGlucose DLactose DMaltose DMannose DRhamnose DRaffnose DSorbtol DXylose Dulctol Nasuccnate Sucrose Acfcaton of meum contanng the followng ntrogen sources: Ca(NO) (NH)S (NH&P (NH)M Alkalnzaton of meum contanng the followng amno acs: L Ar g n n e LHstne LProlne DLSerne DLValne Frst colony forme after: ays ays ays ays ays Growth at: C C C Growth on MPA'. ntrnsc antbotc resstance Ampclln mg/lter mater Kanamycn mg/lter mg/lter mg/lter Penclln mg/lter mg/lter Rfampn mg/lter mg/lter mg/lter Streptomycn mg/lter mgllter mlter NaCl tolerance.%.%.% Reacton wth ltmus mlk Meum alkalnty L L Serum zone Utlzaton of the followng amno acs as sole ntrogen an carbon sources: L Argnne L Contnue on followng page

6 ~~ VOL., NUMRCAL TAXONOMY OF TMPRATZON RHZOBUM STRANS Characterstc LHstne LProlne DLSerne DLValne Utlzaton of the followng carbon sources: D Arabnose DLactose DMannose DRhamnose DXylose TABL Contnue Cluster (n) l() () () () () () () () () () () () () () a, all strans were postve;, all strans were negatve. Numercal values are the number of postve strans of the total number of strans teste. MPA, meatpeptone agar. Three clusters (clusters,, an ) forme the frst taxonomc group of temperatezone rhzoba, wth a. level of smlarty. The central strans of ths group were CUM an CAM. Cluster consste of sx strans solate from root noules forme on temperatezone legumes of varous genera (Lotus, Anthylls, Glycyrrhza, Astragalus, Onons, an Coronlla). Cluster was the most extensve one an contane strans solate from verse legumes from fferent geographcal regons. Cluster, borerng cluster, nclue fve strans specfc for noulatng Heysarum alpnurn an Coronlla vara. Four clusters (clusters,,, an ) mae up the secon taxonomcal group, wth a. level of smlarty. Ths group nclue only strans solate from the root noules of Astragalus an Oxytrops plants foun n Sbera. The central stran of ths group s CAM. The characterstcs that fferentate these newly scovere phenotypc groups from exstng Rhzobum speces are presente n Table. The strans uner nvestgaton were attrbute to the genus Rhzobum (they grew raply on YM mea, not prouce alkal on sugar mea, an utlze a we range of carbon compouns [lo]), whle for most of the characterstcs stue they ha features ntermeate between those the fast an slowgrowng root noule bactera. Data on the plant host ranges of representatve strans of group as well as bactera solate from root noules of C. vara (group ) are presente n Table. Host specfctes of other temperatezone rhzoba nclue n group have been escrbe prevously (). The results show that the plant host ranges of these rhzobal strans of fferent temperatezone geographcal orgns are smlar an nclue several temperatezone legumes (Table ), but no noules were forme on roots of Mecago satva, Trfolum pratense, or Galega orentals plants noculate wth these strans. DSCUSSON The famly Legumnosae, one of the largest plant famles, contans about, fferent speces strbute from arctc to tropcal lattues. Only a small porton of legume speces (less than %) have been screene for ablty to be noulate (, ). Therefore, many bacteral legume mcrosymbonts reman unnvestgate. n recent years, progress towars the evelopment of a more complete Rhzobum taxonomy has been mae, an new speces have been escrbe: R. tropc (), R. huaku (), an Rhzobum etl (). Genomc relateness between members of the famly Rhzobaceae has been reveale by moern approaches an has allowe the constructon of phylogenetc trees an the proposal of possble moes of rhzobal evoluton (,,,, ). The ata gathere to ate show that the famly Rhzobaceae s compose of phylogenetcally heterogenous genera. Thus, Brayrhzoburn spp. are more relate to nonsymbotc bactera such as Rhoopseuomonas, Afpa, an Ntrobacter spp. (, ); Rhzobum spp. are relate to Agrobacterum, Brucella, an Bartonella spp. (). These facts ncate the nee for a revson of exstng taxa of the famly (, ) as well as the lkelhoo of scovery of new forms of root noule bactera. The present work centere on the stuy of root noule bactera solate from the temperatezone legumes of the genera Astragalus, Oxytrops, Glycyrrhza, Onons, Heysarum, an Coronlla. Prevous analyses of the phage senstvtes an plant host ranges of these solates ncate that they are relate to strans solate from root noules forme on Lotus comculatus, an so they were classfe as R. lot (). n ths work, a comparson of the metabolc propertes of the bactera was performe, an cluster analyss was use to construct a enrogram reflectng the phenotypc relatonshps among these strans. A sgnfcant stance was foun between the Rhzobum strans uner nvestgaton an the reference strans nclue n the stuy. n aton, two large groups were entfe among the temperatezone rhzoba stue. Group nclue rhzobal strans orgnatng from fferent geographcal regons of the temperate zone, whle group nclue strans from the same geographcal orgn, South Sbera. None of the rhzobal solates obtane from temperatezone legumes (nclung bactera from L. comculatus noules) were clustere wth the R. lot type stran NZP or wth other R. lot or R. huaku strans. These strans, however, have spectra of phage senstvty smlar to those of the temperatezone rhzoba (ata not presente). t shoul be mentone that taxonomc group nclue stran NZP, whch was solate from the root noules of C. vara. Ths stran was use earler n DNADNA hybrzaton stues an was emonstrate to have not more than % DNA homology wth the R. lot type stran an about % homology wth strans of the R. legumnosarum group (). The present work shows that the Rhzobum strans most closely relate to the temperatezone rhzoba escrbe here were R. legumnosarum strans of the same geographc orgn. Downloae from by P:... On: Tue, Nov ::

7 NOVKOVA T AL. NT. J. SYST. BACTROL. TABL. Metabolc features fferentatng rhzobal specesa Characterstc Acfcaton of meum contanng the followng carbon sources: DArabnose, Dramnose, or Dmannose DXylose Acfcaton of meum contanng mneral ntrogen sources Alkalnzaton of meum contanng the followng amno acs: LHstne LProlne DLSerne Frst colony forme after: ays ays ays Growth on MPA' ntrnsc antbotc resstance Ampclln, mg/lter Kanamycn, mg/lter Penclln, mg/lter Rfampn, mgllter Streptomycn, mg/lter NaCl (.%) tolerance Reacton wth ltmus mlk Acfcaton of meum Serum zone Growth at C Utlzaton of the followng amno acs as sole ntrogen an carbon sources: DLValne DLSerne Utlzaton of the followng carbon sources: DArabnose DXylOSe ', at least % of the strans are postve;, at least % of the strans are negatve;, less than % but more than % of the strans are postve. MPA, meatpeptone agar. The present work also shows that, n general, the numercal classfcaton of the Rhzobum speces use n ths stuy place them nto the same groups as methos base on genomc relateness. Thus, on the bass of ts phenotypc characters, the broahostrange stran NGR was closely relate to the fastgrowng soybean mcrosymbont R. fre USDA (cluster ), consstent wth the results obtane by others from a comparson of s rrna sequences (). The other R. fe stran, whch orgnates from na, was groupe wth the R. mellot strans (cluster ), an ths s also n agreement wth the publshe phylogenetc ata for these two speces (,, ). Strans of R. lot showe a close phenotypc relateness to strans of R. huaku (cluster, exceptng stran CCBAU ), whch also n accorance wth results obtane prevously by usng s rrna sequence comparsons (, ). The type strans of R. lot an R. huaku however, ha no more than % smlarty to the Rhzobum strans solate from L. comculatus noules use n ths analyss. n contrast to the smlartes between the present work an the other work cte above, our enrogram showe that R. tropc CAT was lnke wtha. turnefacens C (bovar ) (cluster ). s rrna sequence analyss ncates that ths stran s more relate to Agrobacterurn sp. bovar or R. Zegurnnosarurn than t s to Agrobacten'urn sp. bovar (,,, ). Nevertheless, the symbotc plasm of CAT can be expresse n an A. tumefacens C genetc backgroun (), an ths fact suggests a egree of phenotypc relateness between these two strans. The metabolc propertes of the R. galegae strans were closely relate to those of Agrobactenum strans (cluster ). The precse taxonomc poston of the R. galegae strans s not yet clear. There s some evence of ther phylogenetc lnkage to R. lot strans (), but other authors showe them to be vergent from R. lot an emonstrate that they were more closely relate to Agrobacten'urn speces (, ). The results of moblzaton of symbotc genes from R. galegae nto Agrobactenum strans woul seem to support the secon fnng (). n any case, formal taxonomc conclusons must be supporte wth ata obtane by fferent systematc approaches. To conclue whether the temperatezone legume Rhzobum strans shoul be assgne to a new speces of the genus or to R. lot wll requre further phylogenetc stuy. Downloae from by P:... On: Tue, Nov ::

8 VOL., NUMRCAL TAXONOMY OF TMPRATZON RHZOBZUM STRANS TABL. Plant host ranges of Rhzobum strans solate from root noules of Astragalus, Oxytrops, an Coronlla plants Noulaton of the followng plant host: Astragalus A A CAM CAM CAM CAM Oxytrops CAM CAM Coronlla CAM CAM NZP a, effectve symboss;, neffectve symboss;, no noules. ACKNOWLDGMNTS We are grateful to Krstna Lnstrom an ntao Wang for knly provng rhzoba strans, Yury Soskov for legume sees, speranza Martnez for encouragng scusson, an Mchael Dunn for reang the manuscrpt. Ths work was supporte by the Research Program nterboazot. RFRNCS. Allen,. N., an. K. Allen.. The Legumnosae: a source book of characterstcs, uses an noulaton. Wsconsn Unversty Press, Mason.. Bouzar, H., an L. W. Moore.. solaton of fferent Agrobacterum bovars from a natural oak savanna an tallgrass prare. Appl. nvron. Mcrobol. :.. Chen, W. X., G. S. L, Y. L. Q,. T. Wang, H. L. Yuan, an J. L. L.. Rhzobum huaku sp. nov. solate from root noules of Astragalus sncus. nt. J. Syst. Bacterol. :.. Crow, V. L., B. D. W. Jarvs, an R. M. Greenwoo.. Deoxyrbonuclec ac homologes among acproucng strans of Rhzobum. nt. J. Syst. Bacterol. :.. arly, B. D., J. P. W. Young, an R S. Selaner.. Phylogenetc poston of Rhzobum sp. stran Or, a symbont of both Mecago satva an Phaseolus vulgars, base on partal sequences of the s rrna an n! genes. Appl. nvron. Mcrobol. :.. lkan, G. H., an C. R. Bunn.. The rhzoba, p.. n A. Balows, H.G. Truper, M. Dworkn, H. Harer, an K.H. Sclefer (e.), The prokaryotes. A hanbook on the bology of bactera: ecophysology, solaton, entfcaton, applcatons, n e., vol.. SprngerVerlag, New York.. Jarvs, B. D. W., H. L. Downer, an J. P. W. Young.. Phylogeny of fastgrowng soybeannoulatng rhzoba supports synonymy of Snorhzobum an Rhzobum an assgnment to Rhzobum fre. nt. J. Syst. Bacterol. :.. Jarvs, B. D. W., M. Glls, an J. De Ley.. ntra an ntergenerc smlartes between the rbosomal rbonuclec ac cstrons of Rhzobum an Brayrhzobum speces an some relate bactera. nt. J. Syst. Bacterol... Jarvs, B. D. W., C.. Pankhurst, an J. J. Patel.. Rhzobum lot, a new speces of legume root noule bactera. nt. J. Syst. Bacterol. :.. Joran, D. C.. Famly : Rhzobaceae Conn, p.. n N. R. Kreg an J. G. Holt (e.), Bergey s mannual of systematc bacterology. vol.. Wllams & Wlkns, Baltmore.. Kreg, N. R.. Systematcs V., p.. n P. Gerhart, R. G.. Murray, R. N. Costlow,. W. Nester, W. A. Woo, N. R. Kreg, an G. B. Phllps (e.), Manual of methos for general bacterology. Amercan Socety for Mcrobology, Washngton, D.C.. Laguerre, G., M. P. Fernanez, V. el, P. Norman, an N. Amarger.. Genomc heterogenty among French Rhzobum strans solate from Phaseolus vulgars L. nt. J. Syst. Bacterol. :.. Lnstrom, K.. Rhzobum galegae, a new speces of legume root noule bactera. nt. J. Syst. Bacterol. :.. Martnez,.. Recent evelopments n Rhzobum taxonomy. Plant Sol :ll.. Martnez,., R. Palacos, an F. Sanchez.. Ntrogenfxng noules nuce by Agrobacterum tumefacens harborng Rhzobum phaseol plasms. J. Bacterol... MartnezRomero,., L. Segova, F. M. Mercante, A. A. Franco, P. Graham, an M. A. Paro.. Rhzobum tropc, a novel speces noulatng Phaseolus vulgars L. beans an Leucaena sp. trees. nt. J. Syst. Bacterol. :.. Novkova, N..,. A. Pavlova, an. V. Lmeshchenko.. Phage senstvty an host range of Rhzobum strans solate from root noules of temperate legumes. Plant Sol :.. Novkova, N.., an V.. Safronova.. Transconjugants of Agrobacterum raobacter harbourng sym genes of Rhzobum galegae can form an effectve symboss wth Mecago satva. FMS Mcrobol. Lett. :.. Orso, S., M. Gouy,. Navarro, an P. Norman.. Molecular phylogenetc analyss of Ntrobacter spp. nt. J. Syst. Bacterol... Otten, L., J. Canaay, J.G. Gerar, P. Fourner, P. Crouzet, an F. Paulus.. voluton of agrobactera an ther T plasms. Mol. Plant Mcrobe nteract... Prevbst, D., L. M. Boreleau, S. CauryReznck, H. M. Schulman, an H. Antoun.. Characterstcs of rhzoba solate from Downloae from by P:... On: Tue, Nov ::

9 NOVKOVA T AL. NT. J. SYST. BACTROL. three legumes ngenous to the Canaan hgh arctc: Astragalus alpnus, Oxytrops mayellana an Oxytrops arctoba. Plant Sol :.. Rnauo, G., S. Orenga, M. P. Fernanez, H. Meugner, an R. Barn.. DNA homologes among members of the genus Azorhzobum an other stem an rootnoulatng bactera solate from the tropcal legume Sesbana rostrata. nt. J. Syst. Bacterol. :.. Sawaa, H., H. ek, H. Oyazu, an S. Matsumoto.. Proposal for rejecton of Agrobacterum tumefacens an revse escrptons for the genus Agrobacterum an for Agrobacterum raobacter an Agrobacten'um rhzogenes. nt. J. Syst. Bacterol. :.. Scholla, M. H.,, an G. H. lkan.. Rhzobumfre sp. nov., a fastgrowng speces that effectvely noulates soybeans. nt. J. Syst. Bacterol. :.. Segova, L., J. P. W. Young, an. MartnezRomero.. Reclassfcaton of Amercan Rhzobum legumnosarum bovar phaseol type strans as Rhzobum etl sp. nov. nt. J. Syst. Bacterol... Sneath, P. H., an R. B. Sokal.. Numercal taxonomy. The prncples an practces of numercal classfcaton. W. H. Freeman & Co., San Francsco.. Trnck, M. J.. Relatonshps amongst the fast growng rhzoba of Lablab purpureus, Luecaena leucocephala, Mmosa spp., Acaca farnesana an Sesbana granflora an ther affntes wth other rhzobal groups. J. Appl. Bacterol. :.. van Rysn, J. Qe.).. Classfcaton an clusterng. Proc. Av. Semn. Math. Res. Center, Unversty of Wsconsn, Mason. Acaemc Press, New York.. Vncent, J. M.. A manual for the practcal stuy of root noule bactera. BP Hanbook no.. Blackwell Scentfc Publcatons, Oxfor.. Welock, D. N., an B. D. W. Jarvs.. DNA homologes between Rhzobum fre, rhzoba that noulate Galega sp., an other Rhzobum an Brayrhzobum speces. nt. J. Syst. Bacterol... Wllems, A., an M. D. Collns.. Phylogenetc analyss of rhzoba an agrobactera base on s rrna gene sequences. nt. J. Syst. Bacterol. :.. Wong, F. Y. K.,. Stackebrant, J. K. Laha, D.. Fleschman, R. A. Date, an J. A. Fruest.. Phylogenetc analyss of Brayrhzobum japoncum an photosynthetc stemnoulatng bactera from Aeschynornene speces grown n separate geographcal regons. Appl. nvron. Mcrobol... Yanag, M., an R. Yamasato.. Phylogenetc analyss of the famly Rhzobaceae an relate bactera by sequencng of s rrna gene usng PCR an DNA sequencer. FMS Mcrobol. Lett. :.. Yelton, M. M., S. S. Yang, S. A. e, an S. T. Lm.. Characterzaton of an effectve salttolerant, fast growng stran of Rhzobum japoncum. J. Gen. Mcrobol. :.. Young, J. P. W.. Phylogenetc classfcaton of ntrogenfxng organsms, p.. n G. Stacey, R. H. Burrs, an H. J. vans (e.), Bologcal ntrogen fxaton. Chapman & Hall, New York.. Young, J. P. W.. Molecular phylogeny of rhzoba an ther relatves, p.. n R. Palacos, J. Mora, an W. Newton (e.), New horzons n ntrogen fxaton. Kluwer Acaemc Publshers, Dorrecht.. Zhang, X., R. Harper, M. Karssto, an K. Lnstrom.. Dversty of Rhzobum bactera solate from the root noules of legumnous trees. nt. J. Syst. Bacterol. :. Downloae from by P:... On: Tue, Nov ::