Application of DNA hybridization to the taxonomy of rhizobium trifolii and related species : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

RHIZO:nu:: TRIFOLII A?rn R~LAT:C:D

SPECI ES

A thes:i s present en in pa.rtial ful f"i.l 1::e:1t for ti1e !'eqn·i rr~::wn t:; fo:::· tb ~: clc_'srcc of t,1:'.tGte:c'a.te of Sci-::-r:ce i:i :-'.:i.crollioJ..-):·v ~v ,:it

Massey Unj_ver;-;j_ty.

Allan Geor~e Dick 1979

A3STnAC'I¹

Rhizob i a

00re

cro~n i

n

Yeast I(a~nit ol

b

roth in the presence and absence o f K 2 HP 0

4•

I ncreasinc the concentra- tion of yeast extrac t in the

mediun

result

ed

in an incr

ease

in gro~th up to a certain concentration of yeast extract . Gro~th of rhizo

bia

~as i nhibited

by

furth

e

r increases in yea

s

t

extra

ct concen tra tion ,

es

pecial ly i n the pre

se

nce of

K

2EPO..,! .

The inh j_bi t ion is

due

to

clyc

ino l)resen t in

t:1e

y

eas'.~ extract ar1d

ir,

L1creased by

t

he

pref,ence o f

::.o

noval

en

t

ea

tio

ns

.

The

~o

l

ec

ular

w

eieht of DNA

extracted

from rhizo~i

a

·::as

detcrnined by cen trj_fu:.:;at j_on :i.n a l kal ine sucrose

₀

ra - dien t s . It ~a

s

found th

at

32 P- l aba lled rn TA ~as more f~a ci l e under shear t han u

:1l

a

be

ll cd D~~A . Unl 2,be ll ed mm

r equir ed

75

seco~ds and

³²

P-l abe ll ed D

N

A r eq~i r e d

56

se con ds son ic ation to r educe the fr a~~en t s ize to

230,000

Dalton s .

Lab elled D ~A prepared by a ph enol -c hlo ro for m meth od

~as co ntam inat e d ~ith polysaccharide and o ~ly lo w ha m q- lo gou s hybridis a tion could be obt aine d. A hyd roxya- pa tit e - urea me thod of purifying D ~A ~as devel uped which produced po ly sa ccha rid e - f re e J ~A. ~he n l abelled D N A w as

·p-,-epo.reJ

72% .

by thi s meth od hoDo lo gou s hy~ r idis&t io ~ avera ged Pol ysa cc haride con taminat io n o f u~la bo lled DNA

preparat io ns did not effect the ~~ r el a tive r eass n~iation.

Co r!l plete reassociation of heterologous DNA required a lo nge r incuba tion time than did .

1

ho m olo ~ous D N A. The homolo gous reac ti on w a s complete after

32

hours (Cot

200)

whe r eas heterolo gous D N A required an i ncubation of 40

hours (Cot 25J) to achieve m axim um reas s ociation.

Decwribonucleic acid ho m olo6ies we re determined

ar:1or..z 27

strains of

Rh·i 7.,0~1i.w1

trj,folii ,

4

stra~.ns

of

F •

J ~

-: u:·11 i nosaru m and 4- of R. uha s eol L ~e su l

ts from

rele,ted strains indicate the.t

DI:tl.

ho

r.:oloc;

ies correlate

~ ith serolocica l r e lation ships and ability t o form nodules on le 5umo roo t s c an

be

lost ~ithou t detectab l e chan~e in llom olo 5y -..·1i th

an

:i.nc.e

r)e

nden t

re ^1. orence

stra.in . A ll rhi- zobia which nodulal:ccl e ff ective ly o n

T . r -2·0c~rs,

T. r:;ubt0rra~1.-::u.rn , _T, .::-,r:~bi•-:u.ur,₂ and

_"\ficjfl

hirsuto.

formed one populat

:1

on l'd.th

,tn

a ver.3..:_:;1; r

e

l atedr.css of 707-;

(ran.::;e L,9-9L~%) ar..d D. I1n ( e)

of

O, 2-7.

5 °

C ':ii

th

re::.; pee

t to r·e fer enc

e

stra in s capable of nodulati nc t~c fir

st

two c lov e r

spscies . Tr:o strains fron

Af.rj_can

.T~i

fnl-i un

c::)ecies and one fro m a J apanese

spec

i

e

s wore l ess

c

lo

se

l y r

e

l ated . The avera,2/? relatedness o f strains fror: 1

?11:::s~oJ.us v1!J,-:1rj_s

·:;it h c l over r hizoj ia ~as 3 7- 50;~ )

a

nd

^D. 'l'r,:(e)

6 . 5-

1

C.

0 0

C. S:axonorr..L c re visions cons i s tent

\'Ii

t h th

cs

c o bs

er-

vati on s are di scu

sGed.

I t i s propo sed that R. trif olii e.;'lcl.

:>, J P[Wii ¹10S(.;.fUi:J

sh oul d be cor:1bined and c a ll e d

1;:~-; ZO'Y; 1:i-¹ l P,'··1;,.•ji oo,;o.::·u1;1

FrcJ.nl~

. \'!i

t i1:.,._i1

ti¹

i s s~1ocies varj_o u. s bi o0p es sho uld be desisnated a ccor din c to t heir plant

spec i ficity R. ~has

c

a li shoul d be re ta in ed as se parate

s p ecies

~nJ

e xam in ed in more deta il. The r esu lts are

discuss ed in rel ation to propo s ed Ge n e tic ba s is

for

plant spe cificity.

ACKNO::.:LE:DGEMEH'l'S

I

am ind.e:':.lted to

the; Dep8.rtn:crnt of -;:;j_cro'oiolo[~Y and

Ge:ietics, 1--:o.r:;sey :Jnj_vcrsi Ly,

for

providj_n:.,'.;

the

oppor-

tu

n

ity and

facilities for thic

investi~ati

on. I

n par

-

ticu

l

ar

I

~ould

like to

thank my supervi

sor, Dr J

arvis

, for

constant

cncoura;_;cnc'.l t

a

nd enthusias

m

.

I ¹::oul

d

al

s

o l i]~e to

t h8.n

k:

the

e~1tj_re

acD.d

err.

ic and t

e

chnical

st

af

f of the

D

e :D ~~tmcnt

of

Ej_cro

:) iol

ocy ancl Ge~1etic[,

for

cencral help

a

nd

providini

friendl

y

s

urrounJinGS

in 0hich to ~ork;

Xr R.

il. Green~ooct, D.S.I.

R.

, for supplying tho

cultures and

help

fu

l

advice

;

Dr

~

.D.

Sut

ton, D.

S

.I .R.,

fo

r

he

l

pful

advice on D

N

A

hyb

r

i

di

sation

;

t·1r J. McLean for

be::.

ng a

steady

source of information;

Mr

R.

Tucker for technical

ass

ist

an

ce a

nd

enthusiasm in the fac

e

of adversity;

Mi

s

s B. Hunter for th

e

typins;

MiRs

J .

Trow for the exc

ellent

dia:rams;

i·~c; C. J. Macdonald, Mr J.B. Smart, and Hiss Y. Sprey

-,

for helpin5 to get it in on time.·

TABLE OI<' CO:i~TENTS

Pae;e ABSTRACT • • • • • • • • • • • • • • • • • • • • • • • • • • i i

ACKNO .

.'/LEDG~MSHTS

. . . . . . . . . . . . . . . . . . . .

LIST OF FIGURES

. . . . . . . . . . . . . . . . . . . . .

LIST OF TABLES

. . . . . . . . . . . . . . . . . . . . . .

I HT~ODJCTIOH

1 • Si6^{ni ficancc of} the Genus :~hizo'oj ^w1

. . . . . . . .

2. Classification By Cross-Inoculation Groups • • •

3 .

Appb.c2.1.'.:,ion of 1;ur:~er icnl 'I'D.xono.::·y and Eol sc ular Geneti cs to the Clac,sificD..tion of Jhi zo':):Lu:'Yj . ••

iv

...

C

5

L1• Detcr r.iination of I3o.so .Sequc~1ce Hor:1olosY • • • • • 11

5 .

Aim of the Invcsti~otion .

. . . . . . . . . .

¹⁵

1

.

2.

1:.ic

ro'oi oloc

j

.. cal i·k

t

hodG

.

• • •

.

• •

. . . . . . . . . . .

1 • 1 1 .2 1

. 3

1 .Ji, 1

. 5

DI:A 2. 1

2.2

f.fodia

. . . . . .

• •

. . . . . . .

•

. .

•

. . . . . . .

i·;:L c

ro

o r

.

:_;;-1-n :Lsr:1c

. . . . . . . .

•

. . .

• •

. . . .

C

ul ti vc,

t

ion

. .

^•

.

• •

. .

•

. .. . . . . . . . . .

•

Gro^{1: :} t

h CU.'.'.''.'

CG

. . . . . . . . .

•

. . . . . .

^•

. .

Cel

l Y

ieJ.cl ExpGriuc

nts

_•

. .

••

. . .

• • •

. . . .

P

repa

r

ation

. . . . . . . . . . . .

• • •

. .

• •

.

•

.

P:\:

para ~

ion

of

U:110.bcl lcd

DI:A

by ^':l.

r~od ifi

co.- t;_on

of

t:rn

H

ctr

.od. of

B

r

e:rnc

r

et o.l.. (19.S9) • 2. 1.1 Lysi.c

of

^Cells

. . . . . . . . .

2. 1 • 2 2. 1 • 3 2. 1.1: ..

2. 1 •

5

2. 1 •

6

~,~traction of D:~1'\ •• •• . • • • Pu.

ri fi

cD- tion

of

^{D:rA • • • • • •}

~out.ine

r:c:::i.s ur

e::;cn

t

^of

PL ·

rj_ty

C

o:icen

tratio n of

^D:Tl\. • • • • • •

Sheo.ril1,S

of

DHA • • •

. . . . . .

. .

•

.

of . .

•

. . .

. .

• •

•

. . .

.

_•

. .

D:'.JA .

. .

•

.

• •

. .

Pr

epo.ra t:Lon of Lo.·oel l ed.

D:rn. • • • • • • • • • • • • •

2.2.1 I,ys

i

s

a

n

d

:Sxtra

ct

io

n of

D~rn . • • • • • • • 2.2.2

P·ce pa ratio

:1

of Eyd

ro

xya pa t

ite ••••••

2. 2 . 3 Hydroxya patit e Purific

a

tioil of DNA

_{• •} 2. 2. L;. Shearin.c.;

of

D:'.fA • • • • • • • • • • • • • • • 2. 2.

5 Strippin.::;. of

^{D?{A • • • •} • • • • • • • • • •

Pac;e

16

16 16

17

1

7

21

22

22 22

23

23 2L: . 2Lf-

2~

2L~

24 24

25 25 26

3 . M

et hods

of A

ssess

ing the Suitabilityof

LJNA for

Hybridisation

Experir.wn ts

• • • • • • • •

• •

• • • • • •

26

3 .1 Determina tion of Meltin5 Temper

a

ture

• • • • • •

3 . 2 3 . 3

of Doubl

e-

Stranded

D:NA • • • • • • • • • • • • • • • 26

Mo

l

ecul ar 'l/eiGht Determinat

i

on

_{•• • • • • • • • •}

Cole

;_1rim

etri c Determination of

DNA

Concentration . . .

• • • • • • •• • • • • • • • • • •

Colourim

e

tric Deterr.1ination of

Protein

Concentration

_{• •• • •}

. . . .

^{• • • •} •• • • • • • •

The Method of Brad ford

( 1976) • • • • •

The Method o

f

Lo

~ry

et al

.

(1 J5 1)

_{• •}

28

29 30 30 30 3 . 5 Colourimetric

D

eter m

ination

of Polysaccharide

Concentration

• • • • • • • • • • • • • • •

..

• • • • •

33 3 . 5 .1 The Method

of·

Trevelyn and Ha

rrison

(1 952 ) . . . • • . . . 32

3 . 5 . 2 The Method of Bailey (1 967 ) •.•••• 32

3 . 6

^{Col ourj_metr}:Lc Deterir,ination of

Ribonucleic Ac\d Cone ci1 L ro. tion.

. . . . . . . . . 33 33 4 .

Basic .Seq ucnce Analyc,ir:i • • • • • • • • • • • • • • • • • •

33

L; • 2

Reo.ss08j_ a tion of DNA

. . . . . . . .

^{• • e • • •}

3)

Sep~ration of Si~:le and on Hydroxynpatitc •• ••

Double-stranded D~A

. . . . . . .. 37

\ . 3

Thern:al .Stab:Ll i ty of l=:r:!D.C:c;ocia t eel Dupl exes • •

38

L1 .l 1. Stc~tir;¹dc:).J. l.nc,lJsis of ¹."1cat3:.,ocio.t:Lon Data ••

38

( . 5 l=loc;cn0ration or Hydro:-~::,,u::.x'-ti te • • • • . . • • • • 39

R':;sULTS

1. Gro':tth of l~hizobiun • • • • • . • • • • • • • • • • • • • • i+O 1 • 1 Gro·:;th Curves •

..

^{• • • • • • • • • • • • • • L!.0}

1 • 2 Ir-1provemon ts in Cel l Yj_eld • • , • • • • • • • • • LrO

2. I-Iol ecul ar ·:tei cht Dotornination of DNA • • • • • • • •

~5

3 .

2. 1 lfolecular 'Jeic;ht of Ur..soni cated D1':A

. . . .

• • 2.2 l·1ol ecular \'/ ei ,:_:;r1

t

of Sonj_cated D:rA

.. . . . . . .

Difficulties Encountered in Hybrj_dif.:;atj :.m

.. . .

^• •

3 .

1 ^First

3 .

1 • 1

3 .

1 • 2

3 .

¹

. 3

Preparation of l abelled DI'TA

. . . .

• •

..

At tenl)t eel Eybridisa tion • • • • • • • • Li-5

Absorbance Spee trum • • • • • • • • • • • • L+8 Ther~al Mel t ini Curve of ~eassociated DlTA • • • • • • • • • • • • • • • • ~ • • • • • •

48

Separation of DHA • • • • • • • • • • • • • • Lr8 Assay of Potential Conta~i nants in

Labelled and Unl abelled ff{A • • , • • • •

49 3.2

Second Preparation of Lbbelled DNA • • • • • • •

49

3 .

2. 1 3.2.2

3 .2. 3

j .2.~-

Absor ba:nce spec. tr_dl .and At tempted

Hybridisation • • • • • • • • • • • • • • ^{er • •} 1:-9 Assay of Potential Contamina~ts in

the Second Preparation of labelled m·A

51

Stripping of unlabelled DNA • • • • • • •

51

Ult~a cen

32

^ifu0ation of Sonciated and Stripped -P-DNA • • • • • . • • • . • • • • • •

51

4 .

3 . 3

^{Puri fico. tion o; D~,f A by tli 0 Hydrnx.yo.-}

pa. U. l:r-;-TJ rea Pcocedure •. •. • . . • ••

. . . . . .

.., 3 ? :J • • ·-

3

.

^{:; ·1.}

.

^{I )·}

Purific:::t:ion of 0:,rt1. Pr0.!'i."t:'..'Ccl. by U18 PheJ1ol- ':::, 1 oro for·::; P roe <;el.ti r0 • • • • • Prepacuti..on of D':A· by i:hc ::ydroxy:i₁)a--

titc-L: r·¹)G Pr'OCCl~Uro • • • • • • • • • • • • •

Assr1.y of 8')nto.·,.·i.:,Dn t,:; j_n T")"f'.. :?rt;porcd by t:1.c Eydroxv::n_,:1:-.·;tc.,.lJrc';,1 Pi'Occdurc • Su· i· t,..,t 1· 1 ··Lt v f'xo 1)· · :' -<-or c.r·,, > r~ d ·i c-;J. [· i o·r1

,. r.,,i. J -- - "oJ ... - !i. I. .;.JJ ~ .... J ,_ ... ~-

r: ·.;

. / ~

:._,.,.

56

57

3 .ll-• IIybridisat-,i.on Co.nd:i.tio.,:^{0; • • • • • • • • • • • • • •} 57

3 .

^{'-i .;~}

3 ,, ·' ^{• t,-. J}

The

::-:r

^.f,,r,t of p:,o-rih0.t0 \.:. ffc!" !-'.01.,c-¹ 2·t

t y

O^Y\ : 1 t·1~ ' ·~ , ) r r"n ... ~ l ,.,,. _ ,-("·1"#; C.. \, o · ""'I • ~ , • ^... L ,.) ''J . . . • .,~ _,. ., L • , -, <.' ••• r"'rr, < D"t11 ¹,._, __ 1l i,·; --

,Strrtnd·.:cl J1.;i\ ~:1:Ll:;1 Iiy(;_Pc>·-'~/·;r,t":.t:Lt2 • • • •

~ffocr, nf Cot ^(Y: Jr:J. lc·,~,·::·r)ci,"Jt:Lon ••

::::xpc:;:··i_·:.cn ts to Jr;crec.,_so '·.;1e: Ap[):\r<::nt

Self-:\,::.::.ociati o:1 of la';,,:,, led. D~TA • • • •

62

mm

HoJ.1010::;y Arr:on:._; ,') train~:; of '.:)

C , '

t-. r·i fnl i,i and Rela terl · ,Jp ec l e.:-""J • • • • • • • • • • • • •

• • - • $ • • • • • • • • •

63

1+. 3

L,. • 4

Labelled DNA Pr3parations

. . . . . . . . . . . .

L;. • 1 • 1

4 .

1 • 2

S p 9 C i f j_ C ti_ C t :i. V j t 0 S • • • • • • • • • • • •

HybridiGation-Self Association of

L<J. be1·1..

1:;o.

D:-~ A, Eor::olo:_;ou:~ ~cac tion, and Pr epar:.,tion of Unlabell ed D:!A • • • • • • Therr,:o.l Stability of ff.~~\. !ieo.::;soc"La t8d at

G 5"c.

Lj .2.1 T-hermal cl.utio~1 profil es •·. • • Li . 2. 2 TherrnGl 5indiT:.:,; In die es ..

Homolocies A1,1on.::; Rel ated Strains

. . ..

• •

L,.3.1 Se:rol osi.cal ly ~~lated S tr0.ins _••

. . . .

4 .3 . 2

^{Hon-:<:-ouula tin}e; Variants

..

^{• •}

. . . .

.

^.,

..

³

'j'. ^{_.J. Different Historj_es of Reisol at es}

. . .

4 . 3 . 4

^{Same Host O}ri 6i n ...

.... . . . . . .

• •

..

DNA Homol o 6^y and Plant Speci fie ly

. .

• • •• ••

63

64

61.i.

61 G 7 67

71 71 71 , 76

4 . 4 .1

^{Horeol ogi cs A}~onB the European Clover

Rhizo bia • • • • • • • • • • • • • • • • • • 76 4.4.2 Homol o:3ies Bet\':eerr' R. 10,-q,-1j riosarum

and the Suropean Cl over 2hizobia ••••

76 4

^.1+.

3 . ·

·Homolot:;i es BeVvcen Ai:1erican Clover

~hizobia and the European Clover

Rhizo bia . • • • • • • • • • . • • • • • • • • 76

4. 4. 4

Homol osies Between Trj_folium

amb j cuum

Rhizobia and European Clover Rhizobia

77 4 . /~ . 5

Homoloc;i es Between African Clover

Rhizobia and European Clover Rhizobia

77 4 . 4 . 6

^{Homol osies Between Japanese Clover}

Rhizo'i):Lurn and :SUi~opean Clover :C::hizobia 81

L1 .•

4 . 7

Homoloc;ies Bet\.\'een

E?b

i z,o h i 11¹n Pha r; col i

and Eurpeean Clover Ri izooit1 • • • • • • 81

L1 .• l~.8 Homolo.:.;ies Bet\'JCGn Rh~_zo'oiun Phascol i and other eroups of ~hizobia • • • • • • 82

DI

SCUSSI

O

N

1

. Gro

¹

:1th

^{of m1i7.0 bi. um}

. .

• • 1 • 1 Gro\·fth

Curv

es

. . . .

1 .2

I

rnpro v er11en t::-, in

C

ell

• •

. . . . . .

. . . . .

•

..

Y:L

e:lu

.. . .

•

. . . . .

. . . . ..

•

.

.

•

. .

Pa

ge 85 85

86

2. Mo

l

e

cul

ar ~eiGht Determination of DNA .. •• •• ..

8 7

-z,

_.J • . Difficulties Encountered in Hybridi sation • 88

3 . 1 Pr

eparati

on of DNA . . . . . . . .

88

3 .

^{1 • 1}

3 .

^{1 • 2}

3 .

^{1 • 1}

?henol-c:110:coforn ::0thod • • • • • • • . • 88 Hycl.ro:,:y:::1.1):J.tite-Urno.. i·Icthod.

• • • • • •

88 ::ffect of Po

l

ysaccho..rj_de ir: th0

Lab

elle

d D:rn

Prepo.ration on Eybrj_c]j_"ation

• • • •

89

3 . 2

Opti~ioation

of

Hybridisation

. . . . . . . . . . 90

3.2.1 ?ffective Phospha te, 3u

ff

er ?-ioJo.r.i..ty on L,e Sepc,·~·c1.tj_on of ,Sj_ncl e 8:r..c; I),')ub

l

c

.S~.randed D~;A ·::i -::h EydroxyD:[).'3.titc) •• ••

90 3 . 2 . 2

Effect of Cot on the ReaEsocio..-~ion of

D:{A • • • • • • . • • • • • . • • • . . • . • • 91

3 . 2 . 3 Ze ro -

ti~e Bindins

of D

enatured

Labe ll

ed

ff,;A to

Hyd

ro

:xyapo.ti

te • • • • • • • • • • • 92

1~. DXA

Ho

mo

lo

cy Am

ong Strains

of ~tizobiu0 trifoli

i

and r

el a

t

ed spe·-:: i es

• • • • • • • • • • • • • . • • • • • •

93

4 .

1

I+. 2

Th

erma

l Staoility o f mm Reas

s

o. ci

o..

te

c.

at

65

°C R

e

l

ationsh

iJS Be t

~een Speci

fic Pairs of

Strains . . . . . . . . . . .

4 - .

2. ^·t

Li.-. 2. 2

4 . 2. 3

I

fon - nod.ula tin

s

variants • • • • • • • • • Ser

ol osi

cally ~

elated

Str

a

ins • • • • • riistorica l Asp

ects

• • • • • • • • • • • •

Ho

rr.

ology and Pl an t Specifici t y . • • • • • • • •

I -,

4· .) .

1

L1 .•

3 . 2

4 . 3 . 3 4 . 3 . 4 4 . 3 . 5 4 .3. 6

Re l a~ionshipa

~ithin Sub

-

gro

ups o f

:R. trj_folji . . . . . : . . . . • . •• •• ••

Relationship Bet

ween R.

Le

,~

u:;1 ino

sa

rur:1·

and the European Clover

~hizobia

•••

·

Re l ationships Bet~

een

B , Ph

as

eoli and Europe~n Clov

e

r

Rhiz

obia • • • • • • • • Propo

sed

C lassification of the F ast - Gr ov:in

e,

Rhizo bia • • • • • • • • • • • • • • Evolutionary C on

s

iderations

.

• • • • • • Pl asmi ds in

B¹i-i zabi nm • • • • • • • • • •

BI B LIO GRAPHY

_{• • • • • • • • • • • • • • •}

•

• • • • • • • • • •

93

91+

94 95 95 96

96 97 98 99

101 103

1 OL~

LIST O

F FIGURES

1. A~paratus for the preparation of linear

suc-'ose :_;radj_ents

. . . . . . . . . . . . . . . . . . . . . .

²⁷

2 .

Colourimetric determinution of

D N A

concentration

by the net hod of ?iurton ( 195::;) • • • • • • •

. . . . . .

³¹

Colourir::etrj_c c1otsTr:i:Ln:ltion of 1·ycotein,

a ) by the 1:1ethod of Brci.dford (19?'~) • • • • •• •• 3l:-

b ) by the r:: o t hod o f Lor: r y et al • ( 1

9 7 ::; ) • • • • • ;,

! ~

Colou.!'.'imetd.c detern:ina tion of r,olysac;charide cone e~1

t

!~at ion,

a) by the ?·iethod of Trevelyn .sind Har:r.·i.son ( 1952)

35

b ) by t h e ^:·1i e t h 0 d o f Bn i l e y ( 1

9 S 7 ) • • • • • • • • 3 5

Colourirr.etric detcrr.1i:1ation of ~:i·A co~1centra tion by the r:1ethod of Ashwel l ( 1957) • • • • • • • • • • • • • • 36

G.

Gro·:1,ch of Jr!-;7,obh1.rn j_n yeast n3.nnitol broth ,•.:j_th

~~d CTithout :inotassiuLl Hvdroren Phosnhatc

~) '.=1:1i7.~i)~ 1:_~.

t--~ ;

^{fol i v;train TA1 • : ••}

. . . . . .

^{L,. 1}

b) Rl1:L7.o:)iu:·1 trj_foJj_ st:..·o.in 1.::U290. • •

.. . . . .

^{I+ 1}

c) Rr,izor)"~m' nhaf;ooli strain TA101 ••

. .

• •

..

^{Lr 1}

7. Effect of incrcasi ns yeast extract concentration f ^{D' . , .} t . ^~ l . . ^{i.. •} 1 / '

on the ;c:ro1·1tb o , .. nizo.J1u:·n r::.1.0. l l sl,ro.Jns . o,

\'!U290, a·nd ~hj_zobi.ur;' :Jho.seol i strain TA101 • • • • • L~3 8. Effect of different batches of yeast extract on the

sro\·1th of ^]J1 iz Obi U':l t:!.~j_ fol ii ,; train 'l'Aj:

a) as rnear;ured l)-:'.~ c^v· ellular pro tej_n

. . . . . . . .

⁴¹⁺

b) as measured by cell dry '•'icicht. _{• •}

.. . . ..

^l1.L1.

9 .

The effect of probe diamet er on the sonication of

DNA. • • • • • • • • • • • • • • • • • , • • • • • • • • • • L1_£

10. The effect of ~?P-labellins on the mol.,cular v:ej_sht of · sonicated DXA • • • • • • • • • • • • • • • • • • • •

~7

11 • Viel tine; curve of incubated homologous DNA from Rhizobiu::-i tri folii strain cc275e. • • • • • • • • • • • • 12. Ultracentrifucat ion of soni cated and stripped

32

_P-

D:NA from RhizobiuE1 t ri fol j j strain ·~c275e.

13. Effect of molar concentrat ion of sodium phosphat e buff.er (pH 6.8) containing 0.4% sodium l auryl_.::.

iul phate on the separat i on of homologous reassocia-

t ed o."'4- l abel led unre2.ssc,ciated DNA as deterr1ined

5.0

by the hydroxyap~ti te batch centrifueation met hod.

60

1 L1-. Effect of oquj_val enl Cot vo..1ue on the reasso- cj_atj_on of :1orr:olo::.!:Ol~S and het r)rolot;ous D:·TA w-J th reference Dl~A extracted from Rhi7,ohi.ur!l

.tri.folij_ strain TA1 • • • • • • • • • • • • • • • • • • • • • 61 15. Therirrc:1.l e1ution lffOfilcs of rcassocjatcd DNA

c!_u.pl.exos - formed at 65°C bet¹:.'0Gn D:';A froi1i

:'?l1i_zo:]i ^l.J"" t r·i folli r3train TA1 and D~:!\ fror:1 other strnins of :.:fr,~_zobi_11~1 trj_folij_.

. . . . . . . .

16. T11er'.:1a1 e1ution profil os of r•Jasr3ociotod DNA

(l.'u"'-~..J l \:j -^0vJ ~ ^ec •• J ' fO"'"'Jr>d ~ l1 .._, ·. r_:..i.. ,.,,,. , ~r::l....1 ./ Or< V ' 1.)P.L"'CCI • .,, l, ',. ''Yl TY· · ;._/ ~-·, ,.i. 1~ '[~rOl'1 .

~l-: ⁱ zo:-i:iur~ :~1:.:"'..s⁰ 0:1_; stro..in cc51 1 and J):'TA fro:n

66

::,tr0.:LI1.S o? J . D!121.soolj_ and '.-1. trj folj i . . • . . . 68

1

7 .

1 8.

1

9 .

20.

The r el ationship bet~een hybridisatio~ ut

65 °C

e-nd. the c.iffe.:'.'ence in nel tinc poi r_t v:l1'ri(e)) of r ocJ.sso ~ Li. t ed. ho !:1010 .::;o us and l~ c t e ::-ol o .'.:;O 1 ;_,;*:-?11 :i.:,, or) i. urn t~~ifol ij_ ::~train TA1 and R. -:-,hn,-ooJt c--:lrc..1.incc.5i1 s.r( D:;A fl~on 11, :?h00;olj_, '.¹ J 0·T'-:ii·~10~:a>:ur:: and

~"1. trifolii • • • • • • • • • • • • • • • • • • • • • • • • 69

-~ Jv-p/E'l<:eS -.fc,-"'~d ^b~tweevi re~.~""'-"- DNA, fro."' ::?cla tionr.,l:j_ n bet:-:ieen thermal hindin,: indic cs

(TSI) and the differ ence in me1ti nc~point

(Ll'I'n(c) ) of re2.ssociD.ted ho:-:,olo:::;ous 8.i:.d het ero- lo,sous du~Jl r;xec formed bet¹:18cn :-eferonco D:~A fror:1 ·*-.

strai:1s of 2. ~Jho.'.'i0()J.i , ~. Je; ·u:.1j_nosP,;¹ ur:1 and

:-? • t rt fol j_ i . . .

70

· *

\<hqol,,.,,""' pl,,,.,~rel, .,+re.,>'\ cc 511 l'.l"'d ON A fy-c.'Y\

Relati onships bet~een the r el ative hyhridisation (~~) at ,S,:,"C of D:;A fron: str8.ins of l~hiz8bia and r efer ence

mu-i.s

fror:1 ~hi7,ob:LU'': tri foJ 4.i strains

cc275e and TA 1 • • • • • • • • • • • • • • • • • • • • • • • • 78 Rel ationship_betITeen the rela tive hybridisation

(% )

at

65 ~C

of DNA frou strains of rhizobia and re fereric e DNAs f'ro1~ ::-frdzo :-:i:;. i1'.-: tri ^foLi_:i_ 2 train

cc275e arid :-foiZG.1):J_·,;i-,1 :)\·Jt~r..r.ioli ;;train cc511 • • • • • 79 21 • ~ela tionship bet\.reen the rela. ti ve hyhridisa tion

(~) at

65° c

of D~A from strains of Hhizobia and reference DITAs from R:1-izob:iu.'.~ Tr"lfoJ-,i strain TA1 and Rhj_zobh:.n uhaseoli strain cc511 • • • • • • • • • • 80 22. Genetic relationship between,str8ins of

~'nizobiun from plants in the· t:;enera Trifol::ium,

Vj cja., Pisur:1 and from Phaseolus vu-l caris . . . 100

LIST OF TABLES

Pa.:;e

I,

^S::_1cc1cs of P1j ^7,nbtwi and thei.r charcJcte-

ri::; Lies ( Af tc:.~ Jorco.'1 and Allen, 1971.r) •• _{• • • •}

II. S_r)c.:cj_es of ~-::, i_7,o·;)i tm o.nd thc"i.r charactc·;~·j_r,tics ( o \; t:t:Lneci. .fro,.: (Jrai1a1•:, 1976; a,; proposed

oy

3

De Le)', 1C)(2:) • • • • • • • • • • • • • • • • • • • • ••

9

III. SL-:-::i.ins of _.,:.; '''):Ji ¹1 • included in tl:ir-, '.:;tucty c1.nd tt1¹;ir D.~1i~_j_t;~., t.o nocl:J.lD.te anti. fix o.tr:1or;i'.1?Gric nitroscn (cffectivc~nss ) on t~o spociGs of

}.1

:·-~~ol.L1-1^{1::. •} • • • • • • • • • • • • • • • • • • • • • 18

IV. A,;so..J's of lo. bol l cd a:1d unlaoAll od DII!'-.. .- pro pared O)' the phenol -ch.loro form r.:ot\10d. • • • • • • • • • •

V .

~ffoct of hydroxyo..patite-urea purification on the abGor:,:).'.1CC :.1:iectro. of t'::o ::;3.ntplcs of DHA

:.·.-.·

... ~ ,_

prepared

oy

the ~1henol -c:-1loro forl'.1 procedure • • • :;~~

VI. Coi.:posi tic11 of frl:'-c t i.oii s o bto. incd in prel i1:1i- rary trialn of the hydroxyapatj_tc-urea pro-

c cdure for D::/\. prepa1~a tion • • • • . • • • , • • • • • 52

VII. Relative hy~ridisation of TA1 reference m!A ~ith ur.lahell ed i)]A prepared by the phenol-chloroform

and the hydroxyapatite-urea procedures •• •• ••

65

VIII. :.1cilo. ti ve r:ybridj_:::;a tion of Dr A strai ns of

?^11-;7,nbi.lJL'l t:r--; fo"J -;, __ · , R. nhasoolj_, .

? . 1,-..,·u·r:i»o:-.J.l'.'Ur,1 a~1d. "S,:c'wrj_·:¹11.a coli \'tith :r,~ fercncc D~~A fro,:. ?.. tri fol-\_j. cc275e at 65ti.C

a;id Cot 100. . . • • • • • • • • • • • • • • • • • • • • • • 72 IX Relative hybridi Gation of DNA from strains of

Rhi ZO:)iun Tri. fol i i .~. nhaseoli. , R. l e·:-u~11i.nosarum

and 3schertc!lia Col i v1ith label led reference D:'.':"A fron ~. trifolii TA1 after reassociation at 65°C

and Cot 250 • • • • • • • • • • • • • • • • • • • • • • • • 7L~

I

X. Relative hybridisation of DXA's fror.i strains . of Rhizobjum trifoJii, 3. p~asAolj,

R.

l e~umi- nosarum, and ~scherichia Coli ~ith reference DNA

from R, nhaseoli cc511 a fter reassociation at

65°C or 80°C at Cot 250 • • • • • • • • • • • • • • • •

83