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Stability of plasmids in Rhizobium phaseoli during culture
STABILITY
Department
OF PLASMIDS DURIKG
of Soil and Crop
IN RHIZOBWM CULTURE
Sciences. Texas A 6: 51 University.
PHASEOLZ
College Station,
TX
77833,
U.S.A.
GRACE R. WEI and D.AVID L. BERRYHILL Department
of !vlicrobiology.
North
Dakota
State University.
Fargo.
ND 55105. U.S.A.
Summary-Genes governing symbiotic nitrogen ftxatton in rhizohia reside on symbiotic (Sym) plasmids. Additional plasmids with unknown functions (“cryptic” plasmids) usually accompany Sym plasmids in rhizobial cells. These cryptic plasmids are assumed to have beneficial roles in the soil environment. Little is known about the maintenance of cryptic plasmids during culture in the laboratory. We have examined the stabilities of both Sym and cryptic plasmids of three Rhi:ohium phustdi strains cultured for 30, 60. 90 and I20 generations. Individual plasmids were labeled with Tn5-Mob. which codes for kanamycin r&stance. to provide an initial means of detecting plasmid loss. Agarose gel-electrophoresis and h,ybridization with Tn.S probe were then used to identify alterations or loss of resistance. Kanamycin-scnstttve isolates were inrrcyucnt and were due IO either losses of TN.S-Moblahcled plasmids or dclctions which included Tn5-Mob DNA. The three Sym plarmids and eight crypttc plasmids studied were highly stable
Plasmids in rhizobia arc of particular intcrcst bccuusc the gcncs coding for nitrogcnase and spcciticity of nodulation occur on sin+ Iargc plasmids called symbiotic (Sym) plasmids m many strains of R/~i:o&on. particui;trly f_ts~growers (Prakash and Athcrly. 1986). tisually. strains newly isolated from ticldgrown legumes contain additional plasmids. The number of plasmids generally ranges from two to four or five, and may be as high as ten (Thurman e’~ trl.. 1985). The numbers and sizes of the plasmids have aided in ditfercntiating rhizobial strains and determining their variability. Little is known about the functions of plasmids other than the plasmid that is involved in nitrogen fixation. However. it is probable that thcsc “cryptic” plasmids have a beneficial role in nature or they would not bc maintained (Shaw. 1957). Plasmid content is expected to remain stable so long as sclectivc pressures provide an advantage to plasmid-bearing cells. The ability of rhizobia to maintain their symbiotic charactcristtcs on culturing is not very dependable; strains often lose some of their effectiveness in nitrogen fixation (Weaver and Wright. 1987). and some even lose their ability to nodulate a particular host. The alterations in symbiotic competence may be due to total loss of the Sym plasmid or to deletions or gcnomic rearrangements (Djordjevic er ul.. 1982: Noel PI al., 1984; SoberonChavez EI ul.. 1986: Flores et cl/.. 1988). It is also important to be aware of the cultural stability of non-Sym plasmids in rhizobia because the information coded in thcsc plasmids may be essential for survival and reproduction in the soil environment. We have examined the stability of Sym plasmids and cryptic plasmids of various sizes during extended
laboratory culture of Rhizohium phcrseoli. Individual plasmids wcrc Iabclcd by transposon mutagcncsis with TnS-Mob clcmcnts that confer kanamycin rcsistancc to cells upon integration in their gcnomes (Simon, I9R-t). Prcsumptivc loss of a labclcd plasmid was indicated by loss of kanamycin rcsistancc and confirmed by agarose-gel elcctrophorcsis.
A~ATEWIAIS
Bircleriul
AND ?vlETHODS
strains
R. phmeali strains I27K I2b. I27K44 and l27K8 I 3 were obtained from The Nitragin Company, Milwaukee, Wis. Each strain (which is nalidixic acid resistant) was mated with the donor Escherichia coli Sl7-I (pSUP501 I) (Simon. 1984); this E. colt’ donor has an RP4 derivative integrated in its chromosome which will mobilize “suicide” vector pSUPSOI I into a variety of gram-negative bacteria. Because pSUP5OI I will not replicate in rhizobia. the TnS-Mob element carried on the plasmid will persist only when it transposes to the rhizobial gcnome. TnS-Moblabeled rhizobial clones were selected on mannitolyeast extract (MYE) agar (Paczkowski and Berryhill, 1979) containing kanamycin (50 pg ml-‘) and nalidixic acid (IS pg ml-t). Only clones in which the various individual plasmids had TnS-Mob insertions (one marked plasmid per clone) were used. Growth conditions Each TnS-Mob-labeled clone was grown in IO ml of MYE broth with kanamycin (50 pg ml-‘) at 30 C, with shaking, until late exponential growth was attained. Then. 0.5 ml of the culture was used to inoculate SO ml of MYE broth (devoid of antibiotics) contained in a 250 ml Erlenmeyer flask. After growth
etrp IO succcssic-c transfers (cu 30 generations for each clone). ceils were plated on SlI’E agrir. and resulting colonies uerc scrccncd for lo55 of kanambcin rcsistancc b> replica plating to XI\r’E ;Ig;Lr containing kanrtmycin.
To contirm the prcscncc and locations of Tn?-.llub inserts, rhizobla aerc subjected to the in-gel ibsis procedure of Eckhardt (1978). essentially as modiiied by Rosenberg t’f ~1. (1952). Following elcctrophoretic separation in the qarosc pcl. the plasmids were partiallv dcpurinated by soakmg the gel in 0.25 M f ICI for 5 min and their DNA WL denatured b> trratmcnt with 0.4 s S:tOH. The plasmid hands wrc elcctroblotted to ;I nylon mcmbranc and hybridized at hl C with a biotinylatcd pBR32::Tn5 probe to detect Tn5-Mob scqucnccs. Probe DiNA u’a labeled with biotin-I I-dUTP by nick translation (Ripby C[ (II.. 1977) and hybridirattons wcrc visualized with the HluGENE detection system (Bcthcsda Rcscarch Laboratorw). The identities of Sym pl;wiids st’rr’ confirm4 in 3 similar m:inncr using ni/ prohc pRmR2 (Ruvkun LPItrl.. IWI’).
at 30 c‘, with shaking. 0.5 ml of lhc rcwlting culture was tran4crrcd to 50 ml 01‘ frc4 Mt’E broth. The doubling timcx of strain\; 137Kl12h. i27K;J-l and 127K;HI-i Hcrc 7.5. X.0 2nd 2.8 h. rcspccli\cl~. r+hile dnubiln~ timor of ~hc Tn5-\loh-l;lhclccI dcrlvxtivcs wrc the wmc ;IX thsir parent<. or varied -C 10”IB from Ihcm. c‘lonc~ dcrircd from Grains I37K I Zh and I27K1-l \\crc trx&2-red c\cry 4X h, \v hilt th0’rc from strain 127KXI-3 wcrc twnsfcrrcd rvcr) 24 Ii. APtcr
The plaw~itl plofilcs of the three p;lrcnt rhi/~>ixll strxirw arc shown in I2.g. I. l’l;wiid si/cs wcrc c5tim;llcd using Ihc IhO and 1-W Md pl;i\mitls of /i p/f~~sw/i 111sI as standard (Ilt;lst03on (‘I irl.. I OS?) 'l'hcSym plxsmids Hcrc itlwtilicd by hydridwation with the hiotin>l;ltctl rri/‘prc)hc; their si/cs in strains I27K I%, 117K44 and 127KXI-3 xc 250, X5 ;md 210 Xld, rqcctivcl>.
sue of
f27Rl’b 4: t27E-U 27 IXKU 29 l27K41-3 J? 127KYl-3 I 12iKSl-3. IO 1?7Kdl-3 I5 I?iE$I-3~?5 IYRRI-3 26
Kanamycin-senrili*e isolales,Xx tested after gcnrolrvn
lnbeled plasmid (&id)
30
460 ?ZY 545 I30 210‘ 2lV 210* 250 sxs
0’254 0 50 0 250 i,250 0-1 0,;7 0125 0,250 0 x2
Rhizobinl strain 127Kl2b has five plasmids ranging in size from 130 to 460 Md (Fig. I). TnS-Mob was inserted into each plasmid (Fig. 2). and, after culturing for 30 or more pcnrrations. kanismycinsensitive coictnics wcrc not dctcctcd for clvncs having TnS-Mob lo~~tcd in the 330. 25% 21% and 4% Md plasmids (Table I ). ffowcvcr. one kamtmycinsensilivc isol;ttc was ohtaintxi after culturing clone I57K 13,’ I9 (I;th&tf in lhc ?95 Xld plasmid) for 90 grncrations (Tahlc I ): the pla~mid prcrfilc of this isolntc (not shown) indicated that the 395 Md pl;~smid had hccn lost.
Strain 127KXl-3 has f0ur plxmrtls that range in six from I30 to 5X5 W (Fig. I ). and clones of this strain in which TnS-?itc>b had inserttd into sir& plasmids u’crc cultural. Three dilTcrcnt clones having TnS-M&iahcIcJ Sym plasmids were studied
60
0250 0.150 0 250 o::_w 0.205 0,150 0.150 O:?SO 0’10
%I
ll0
0 250 0250 0 250 0 250 1,250 o,qo 2,250 0’250 0 250
0 ‘50 0 2-w 0 2%
I 110 0 0 2 n 0
,‘n 250 250 :.Yl 150
(Table Ii bccausc the unlabeled Sym plasmid had been observed to undcrgv a spontaneous deletion (unpl~bli~h~d results). No kanamycinsensitive colonies rest&cd when ctoncs having lab&d 30 or 585 Md plasmids wcrc cultured. However. knnatnyoin-scnsitivc isolates arc obtained for clones having the transposon inserted into the I?0 or 2 IO Md pl;emids (Tahlc I 1. For clone 127KX l-3.43, the k;in;lniyciri-scnsiti~c dcrivattvcs dctcctcd after 30 and IN gcncrations had each lost the Iabclcd I30 Md plasmid (l:ig. 3). The three clones having TnS-Mob inscrtcd into lhc 210 Llrl Sym pl;lsmid yicldcd dill& cnt results; one k;tn;lniycirl-scnsltiv~ colony was idcntitied for clone I77KS I-3 I :iftcr ‘HI gcnsrations, no kanamy~in-sctisitivc dcriv;ttivcs at‘clonc 127tiXI-3! IO ;mtl two k;rn;lrli~~irl-s~t~~tivi~ wcrc’ c’vcr dct~ted. colonies :tppcarcd after hltth 90 and 120 generations for clvnc 117K,U I-2, I5 (Tahlc I). The kxnzmycinscnsitivc isolate for clone 177KX l-3. I did not i0sc its Sym plasmid (Fig. 4). but the Sym pktsmid no longcr hybridixd with the TnS prohc. Similxly, the four kanamycin-scnsitivc dcrivativcs ofclonc 127KYI-3, I5 at Icast p;lrtially rctaincd their Sym plasmids while losing their ubilitics to hybrid& with the Tn5 probe: howcvcr. &Actions in the Sjm plasmids wtxc much more cvidcnt (Fig. 5).
Plasmid stability in rhizobia Rosenberg C., Casse-Delbart F., Dusha I.. David M. and Boucher C. (1982) Megaplasmids in the plant-associated meliloti bacteria Rhkobium Pseudomonas and solanacearum.
Journal
of Bacteriology
150. 402-406.
Ruvkun G. B.. Long S. R.. Meade H. M.. van den Bos C. R. and Ausubel F. M. (1982) ISRml: a Rhizobium melilori insertion sequence that transposes preferentially into nitrogen fixation genes. Journal of Molecular and Applied Generics I, 405418. Shaw P. D. (1987) Plasmid ecologv. In Pkmr-Microbe Inferactions, Vol2 (T. Kosuge and E. W. Nester. Eds). pp. 339. Macmillan, New York. Simon R. (1984) High frequency mobilization of gram-negative bacterial replicons by the in rirro constructed TnS-
469
Mob transposon. Molecular and General Generics 1%. 413-420. Soberon-Chavez G., Najera R.. Olivera H. and Segovia L. (1986) Genetic rearrangments of a Rhizobium phaseoli symbiotic plasmid. Journal of Eacteriolog_v 167, 487-49 I. Thurman N. P.. Lewis D. M. and Jones D. G. (1985) The relationship of plasmid number to growth, acid tolerance and symbiotic efficiency in isolates-of Rhizobium frifolii. Journal
of Aoolied
Bacrerioloev
58. l-6.
Weaver R: WI and Wright-S. F. (1987) Variability in effectiveness of rhizobia during culture and in nodules. Applied and Encironmentul Microbio1og.v 53. 2972-2974.
Department
OF PLASMIDS DURIKG
of Soil and Crop
IN RHIZOBWM CULTURE
Sciences. Texas A 6: 51 University.
PHASEOLZ
College Station,
TX
77833,
U.S.A.
GRACE R. WEI and D.AVID L. BERRYHILL Department
of !vlicrobiology.
North
Dakota
State University.
Fargo.
ND 55105. U.S.A.
Summary-Genes governing symbiotic nitrogen ftxatton in rhizohia reside on symbiotic (Sym) plasmids. Additional plasmids with unknown functions (“cryptic” plasmids) usually accompany Sym plasmids in rhizobial cells. These cryptic plasmids are assumed to have beneficial roles in the soil environment. Little is known about the maintenance of cryptic plasmids during culture in the laboratory. We have examined the stabilities of both Sym and cryptic plasmids of three Rhi:ohium phustdi strains cultured for 30, 60. 90 and I20 generations. Individual plasmids were labeled with Tn5-Mob. which codes for kanamycin r&stance. to provide an initial means of detecting plasmid loss. Agarose gel-electrophoresis and h,ybridization with Tn.S probe were then used to identify alterations or loss of resistance. Kanamycin-scnstttve isolates were inrrcyucnt and were due IO either losses of TN.S-Moblahcled plasmids or dclctions which included Tn5-Mob DNA. The three Sym plarmids and eight crypttc plasmids studied were highly stable
Plasmids in rhizobia arc of particular intcrcst bccuusc the gcncs coding for nitrogcnase and spcciticity of nodulation occur on sin+ Iargc plasmids called symbiotic (Sym) plasmids m many strains of R/~i:o&on. particui;trly f_ts~growers (Prakash and Athcrly. 1986). tisually. strains newly isolated from ticldgrown legumes contain additional plasmids. The number of plasmids generally ranges from two to four or five, and may be as high as ten (Thurman e’~ trl.. 1985). The numbers and sizes of the plasmids have aided in ditfercntiating rhizobial strains and determining their variability. Little is known about the functions of plasmids other than the plasmid that is involved in nitrogen fixation. However. it is probable that thcsc “cryptic” plasmids have a beneficial role in nature or they would not bc maintained (Shaw. 1957). Plasmid content is expected to remain stable so long as sclectivc pressures provide an advantage to plasmid-bearing cells. The ability of rhizobia to maintain their symbiotic charactcristtcs on culturing is not very dependable; strains often lose some of their effectiveness in nitrogen fixation (Weaver and Wright. 1987). and some even lose their ability to nodulate a particular host. The alterations in symbiotic competence may be due to total loss of the Sym plasmid or to deletions or gcnomic rearrangements (Djordjevic er ul.. 1982: Noel PI al., 1984; SoberonChavez EI ul.. 1986: Flores et cl/.. 1988). It is also important to be aware of the cultural stability of non-Sym plasmids in rhizobia because the information coded in thcsc plasmids may be essential for survival and reproduction in the soil environment. We have examined the stability of Sym plasmids and cryptic plasmids of various sizes during extended
laboratory culture of Rhizohium phcrseoli. Individual plasmids wcrc Iabclcd by transposon mutagcncsis with TnS-Mob clcmcnts that confer kanamycin rcsistancc to cells upon integration in their gcnomes (Simon, I9R-t). Prcsumptivc loss of a labclcd plasmid was indicated by loss of kanamycin rcsistancc and confirmed by agarose-gel elcctrophorcsis.
A~ATEWIAIS
Bircleriul
AND ?vlETHODS
strains
R. phmeali strains I27K I2b. I27K44 and l27K8 I 3 were obtained from The Nitragin Company, Milwaukee, Wis. Each strain (which is nalidixic acid resistant) was mated with the donor Escherichia coli Sl7-I (pSUP501 I) (Simon. 1984); this E. colt’ donor has an RP4 derivative integrated in its chromosome which will mobilize “suicide” vector pSUPSOI I into a variety of gram-negative bacteria. Because pSUP5OI I will not replicate in rhizobia. the TnS-Mob element carried on the plasmid will persist only when it transposes to the rhizobial gcnome. TnS-Moblabeled rhizobial clones were selected on mannitolyeast extract (MYE) agar (Paczkowski and Berryhill, 1979) containing kanamycin (50 pg ml-‘) and nalidixic acid (IS pg ml-t). Only clones in which the various individual plasmids had TnS-Mob insertions (one marked plasmid per clone) were used. Growth conditions Each TnS-Mob-labeled clone was grown in IO ml of MYE broth with kanamycin (50 pg ml-‘) at 30 C, with shaking, until late exponential growth was attained. Then. 0.5 ml of the culture was used to inoculate SO ml of MYE broth (devoid of antibiotics) contained in a 250 ml Erlenmeyer flask. After growth
etrp IO succcssic-c transfers (cu 30 generations for each clone). ceils were plated on SlI’E agrir. and resulting colonies uerc scrccncd for lo55 of kanambcin rcsistancc b> replica plating to XI\r’E ;Ig;Lr containing kanrtmycin.
To contirm the prcscncc and locations of Tn?-.llub inserts, rhizobla aerc subjected to the in-gel ibsis procedure of Eckhardt (1978). essentially as modiiied by Rosenberg t’f ~1. (1952). Following elcctrophoretic separation in the qarosc pcl. the plasmids were partiallv dcpurinated by soakmg the gel in 0.25 M f ICI for 5 min and their DNA WL denatured b> trratmcnt with 0.4 s S:tOH. The plasmid hands wrc elcctroblotted to ;I nylon mcmbranc and hybridized at hl C with a biotinylatcd pBR32::Tn5 probe to detect Tn5-Mob scqucnccs. Probe DiNA u’a labeled with biotin-I I-dUTP by nick translation (Ripby C[ (II.. 1977) and hybridirattons wcrc visualized with the HluGENE detection system (Bcthcsda Rcscarch Laboratorw). The identities of Sym pl;wiids st’rr’ confirm4 in 3 similar m:inncr using ni/ prohc pRmR2 (Ruvkun LPItrl.. IWI’).
at 30 c‘, with shaking. 0.5 ml of lhc rcwlting culture was tran4crrcd to 50 ml 01‘ frc4 Mt’E broth. The doubling timcx of strain\; 137Kl12h. i27K;J-l and 127K;HI-i Hcrc 7.5. X.0 2nd 2.8 h. rcspccli\cl~. r+hile dnubiln~ timor of ~hc Tn5-\loh-l;lhclccI dcrlvxtivcs wrc the wmc ;IX thsir parent<. or varied -C 10”IB from Ihcm. c‘lonc~ dcrircd from Grains I37K I Zh and I27K1-l \\crc trx&2-red c\cry 4X h, \v hilt th0’rc from strain 127KXI-3 wcrc twnsfcrrcd rvcr) 24 Ii. APtcr
The plaw~itl plofilcs of the three p;lrcnt rhi/~>ixll strxirw arc shown in I2.g. I. l’l;wiid si/cs wcrc c5tim;llcd using Ihc IhO and 1-W Md pl;i\mitls of /i p/f~~sw/i 111sI as standard (Ilt;lst03on (‘I irl.. I OS?) 'l'hcSym plxsmids Hcrc itlwtilicd by hydridwation with the hiotin>l;ltctl rri/‘prc)hc; their si/cs in strains I27K I%, 117K44 and 127KXI-3 xc 250, X5 ;md 210 Xld, rqcctivcl>.
sue of
f27Rl’b 4: t27E-U 27 IXKU 29 l27K41-3 J? 127KYl-3 I 12iKSl-3. IO 1?7Kdl-3 I5 I?iE$I-3~?5 IYRRI-3 26
Kanamycin-senrili*e isolales,Xx tested after gcnrolrvn
lnbeled plasmid (&id)
30
460 ?ZY 545 I30 210‘ 2lV 210* 250 sxs
0’254 0 50 0 250 i,250 0-1 0,;7 0125 0,250 0 x2
Rhizobinl strain 127Kl2b has five plasmids ranging in size from 130 to 460 Md (Fig. I). TnS-Mob was inserted into each plasmid (Fig. 2). and, after culturing for 30 or more pcnrrations. kanismycinsensitive coictnics wcrc not dctcctcd for clvncs having TnS-Mob lo~~tcd in the 330. 25% 21% and 4% Md plasmids (Table I ). ffowcvcr. one kamtmycinsensilivc isol;ttc was ohtaintxi after culturing clone I57K 13,’ I9 (I;th&tf in lhc ?95 Xld plasmid) for 90 grncrations (Tahlc I ): the pla~mid prcrfilc of this isolntc (not shown) indicated that the 395 Md pl;~smid had hccn lost.
Strain 127KXl-3 has f0ur plxmrtls that range in six from I30 to 5X5 W (Fig. I ). and clones of this strain in which TnS-?itc>b had inserttd into sir& plasmids u’crc cultural. Three dilTcrcnt clones having TnS-M&iahcIcJ Sym plasmids were studied
60
0250 0.150 0 250 o::_w 0.205 0,150 0.150 O:?SO 0’10
%I
ll0
0 250 0250 0 250 0 250 1,250 o,qo 2,250 0’250 0 250
0 ‘50 0 2-w 0 2%
I 110 0 0 2 n 0
,‘n 250 250 :.Yl 150
(Table Ii bccausc the unlabeled Sym plasmid had been observed to undcrgv a spontaneous deletion (unpl~bli~h~d results). No kanamycinsensitive colonies rest&cd when ctoncs having lab&d 30 or 585 Md plasmids wcrc cultured. However. knnatnyoin-scnsitivc isolates arc obtained for clones having the transposon inserted into the I?0 or 2 IO Md pl;emids (Tahlc I 1. For clone 127KX l-3.43, the k;in;lniyciri-scnsiti~c dcrivattvcs dctcctcd after 30 and IN gcncrations had each lost the Iabclcd I30 Md plasmid (l:ig. 3). The three clones having TnS-Mob inscrtcd into lhc 210 Llrl Sym pl;lsmid yicldcd dill& cnt results; one k;tn;lniycirl-scnsltiv~ colony was idcntitied for clone I77KS I-3 I :iftcr ‘HI gcnsrations, no kanamy~in-sctisitivc dcriv;ttivcs at‘clonc 127tiXI-3! IO ;mtl two k;rn;lrli~~irl-s~t~~tivi~ wcrc’ c’vcr dct~ted. colonies :tppcarcd after hltth 90 and 120 generations for clvnc 117K,U I-2, I5 (Tahlc I). The kxnzmycinscnsitivc isolate for clone 177KX l-3. I did not i0sc its Sym plasmid (Fig. 4). but the Sym pktsmid no longcr hybridixd with the TnS prohc. Similxly, the four kanamycin-scnsitivc dcrivativcs ofclonc 127KYI-3, I5 at Icast p;lrtially rctaincd their Sym plasmids while losing their ubilitics to hybrid& with the Tn5 probe: howcvcr. &Actions in the Sjm plasmids wtxc much more cvidcnt (Fig. 5).
Plasmid stability in rhizobia Rosenberg C., Casse-Delbart F., Dusha I.. David M. and Boucher C. (1982) Megaplasmids in the plant-associated meliloti bacteria Rhkobium Pseudomonas and solanacearum.
Journal
of Bacteriology
150. 402-406.
Ruvkun G. B.. Long S. R.. Meade H. M.. van den Bos C. R. and Ausubel F. M. (1982) ISRml: a Rhizobium melilori insertion sequence that transposes preferentially into nitrogen fixation genes. Journal of Molecular and Applied Generics I, 405418. Shaw P. D. (1987) Plasmid ecologv. In Pkmr-Microbe Inferactions, Vol2 (T. Kosuge and E. W. Nester. Eds). pp. 339. Macmillan, New York. Simon R. (1984) High frequency mobilization of gram-negative bacterial replicons by the in rirro constructed TnS-
469
Mob transposon. Molecular and General Generics 1%. 413-420. Soberon-Chavez G., Najera R.. Olivera H. and Segovia L. (1986) Genetic rearrangments of a Rhizobium phaseoli symbiotic plasmid. Journal of Eacteriolog_v 167, 487-49 I. Thurman N. P.. Lewis D. M. and Jones D. G. (1985) The relationship of plasmid number to growth, acid tolerance and symbiotic efficiency in isolates-of Rhizobium frifolii. Journal
of Aoolied
Bacrerioloev
58. l-6.
Weaver R: WI and Wright-S. F. (1987) Variability in effectiveness of rhizobia during culture and in nodules. Applied and Encironmentul Microbio1og.v 53. 2972-2974.