P l a n t a n d Soi l XXVI , n o .

1 F e b r u a r y 1967
ME T HODS OF DE T E CT I ON AND E S T I MAT I ON OF
RHI Z OBI A I N S OI L
b y J. A. THOMPSON* and J. M. VI NCENT* *
Mi crobi ol ogy La bor a t or y, School f Agr i cul t ur e, Uni ve r s i t y of Sydrl ey,
Sydne y, New Sout h Wal es, Aus t r a l i a
I NT RODUCT I ON
Det ect i on of specific rhi zobi a in soll, and t hei r quant i t at i ve de-
t ermi nat i on, are essent i al for ecological st udi es ai med at defining
t he fat e of rhi zobi a on seed and seedling, and t hei r persi st ence for
longer peri ods in t he soll and rhizosphere. Such defi ni t i ve isolation
of rhizobia from t he soil, in whi ch at t he best t hey are l i kel y t o
const i t ut e a small and not ver y vi gorous par t of t he t ot al popul at i on,
rest s on t hei r capaci t y t o nodul at e a specific l egumi nous host. Pre-
cision in det ermi ni ng t he number of rhi zobi a will t hen depend on t he
agreement bet ween t he number in t he soil and t he number est i mat ed
b y what is necessari l y an indirect met hod. The principle of this
i ndi r ect ( "pl ant - di l ut i on" or "pl ant i nfect i on") count ( Wi l s o n 13)
has been fol l owed b y ot hers i nt er est ed in t he quant i t at i ve r ecover y
of rhi zobi a from soil and ot her bi ol ogi cal l y compl ex si t u-
at i ons 1 2 6 10 11. Pur e cul t ures of rhi zobi a appear t o offer no part i -
cul ar di ffi cul t y in obt ai ni ng reliable count s b y a pl ant -di l ut i on
t echni que in t ubes. A consi derabl e body of publ i shed 2 and un-
publ i shed dat a from t hi s l abor at or y showed l i t t l e difference be-
t ween t he pl at e and pl ant count s for means based on 50 compari sons
wi t h cl over and 34 wi t h lucerne.
The si t uat i on as t o r ecover y from soil is less clear. I t is difficult t o
i nt er pr et Tuzi mur a and Wat anabe' s findings in t he presence of soil
* Pr e s e nt a ddr e s s : New Sout h Wal es De p a r t me n t of Agr i cul t ur e, Re s e a r c h St at i on,
Ta mwo r t h , N. S. W. , Aus t r al i a.
** Preselxt a ddr e s s : School of Bi ol ogi cal Sci ences, Ul l i ver s i t y of New Sout h Wal es,
Kensi rl gt orl , N. S. W. , Aus t r al i a.
- - 7 2 - -
DETECTI ON AND ESTI MATI ON OF RHI ZOBI A IN SOl L 7 3
because t hei r r ecover y was sub- opt i mal even in its absence 11. A1-
t hough Brockwel l 1 f ound t he pl ant i nfect i on met hod sat i sf act or y
for count i ng rhi zobi a added t o t wo ki nds of soil, t he maxi mum
quant i t y of soil carri ed over wi t h t he l east di l ut ed sampl e was onl y
0.01 g. Larger amount s of soll (1 g o r even more) ma y have t o be
i ncl uded when l ow-count solls are bei ng st udi ed and we cert ai nl y
have had i ndi cat i ons of i nt erference in some such cases ( e . g . l east
di l ut ed, wi t h 1 g soil: negat i ve; more diluted, wi t h 0.1 g or 0.01 g
soil: positive). I n t hese ci rcumst ances it becomes i mpor t ant t o decide
whet her such lower di l ut i on ' ski ps' ( Ph e l p s , quot ed b y Ha l v o r s e n
and Z i e g l e r 5) are t o be accept ed as a source of r andom error,
al r eady t aken i nt o account in " mos t - pr obabl e- number " t abl es, or
whet her such negat i ve t ubes are i ndi cat i ve of non- r andoml y un-
f avour abl e condi t i ons for nodul at i on. The possi bi l i t y of i nt erference
wi t h r ecover y bei ng associ at ed wi t h a r el at i vel y large amount of soil
(1 g o r 0.1 g) added t o t he agar t ube suggest ed a need t o eval uat e
ot her assembl i es in whi ch such effect s mi ght be less mar ked, part i cu-
l ar l y for t he r ecover y of rhi zobi a from spar sel y popul at ed solls.
Wi t h such soils more di rect less quant i t at i ve met hods mi ght t hen
have t o be used.
The following st udi es were i ni t i at ed t o exami ne fact ors governi ng
t he rel i abi l i t y of met hods avai l abl e for det ect i on and est i mat i on of
rhi zobi a from soil.
MATERI ALS AND METHODS
Sol l s
Th e soi l s u s e d i n t h e s e s t u d i e s we r e f r o m t h e o u t e r c u l t i v a t e d b o u n d a r y of
t h e Pi l l i ga r e g i o n n e a r Na r r a b r i , l qe w S o u t h Wa l e s . Th e pr of i l e c o r r e s p o n d s
t o s ol odi z e d s o l e n e t z 4 wi t h s a n d y s u r f a e e soi l s of l ow f e r t i l i t y a n d p H 4. 5- 5. 0.
Ot h e r s t u d i e s 12 h a d s h o wn t h a t e q u a l p a t t s of l i me a n d s u p e r p h o s p h a t e we r e
l l e c e s s a r y f or s uc c e s s f ul e s t a b l i s h me l l t of speci es of Tr i [ ol i um a n d Medi cago
a n d t h e i r r h i z o b i a .
Bi ol ogi cal mat eri al s
Th e s e i l l v e s t i g a t i o n s we r e c ol l c e r ne d wi t h Rhi z obi um tri]olii st r ai i 1 TAl ;
t h e t e s t h o s t i n al l cas es wa s Tr i f ol i um subt erraneum L. Al l s ur f a c e - s t e r i l i z a t i ol l
of s e e d wa s c a r r i e d o u t u s i n g 0 . 2 % me r c u r i c c h l o r i d e s o l u t i o n f ol l owe d b y
r e p e a t e d wa s h i n g s i l l s t e r i l e wa t e t .
Quant i t at i ve, indirect, methods
I d e a l l y , p l a l l t - d i l u t i o n c oul l t s de pe l l d o n l l o d u l a t i o n wi t h as f ew as ort e
v i a b l e r h i z o b i a l cel l p e r p l a n t a s s e mb l y ul l i t . Co n d i t i o n s t a u s t , t he r e f or e , b e
74 J . A. THOMPSON AND J . M. VI NCENT
s a t i s f a c t o r y f o r m u l t i p l i c a t i o n , c o l o n i s a t i o n o f t h e r o o t s u r f a c e a n d f o r m a t i o n
o f n o d u l e s . T h e p u r e c u l t u r e p l a t e c u n t , w i t h c a r e f u l a t t e n t i o n t o u s u a l
p r e e a u t i o n s a s t o m i x i n g a n d u s e o f p i p e t t e , t h e n s e r v e d t o e h e c k t h e t o t a l
s u i t a b i l i t y o f c o n d i t i o n s . I n s o m e c a s e s t h e d i l u t i o n s e r i e s w a s e x t e n d e d t o
p r o v i d e f o r a n e s t i m a t e b a s e d o n p r e s e n c e o r a b s e n c e o f r h i z o b i a l g r o w t h
i n t h e p l a t e ( " P l a t e - e s t i m a t e " ) .
W e h a v e u s e d t w o a s s e m b l i e s f o r p l a n t - d i l u t i o n c o u n t s :
( a ) B o t t l e j a r s . T h i s p a r t i c u l a r b o t t l e j a r a s s e m b l y w a s d e v e i o p e d f r o m
t h a t o f L e o n a r d s 9 ~ p r i m a r i l y a s a m e a n s o f g r o w i n g l a r g e l e g u m e s u n d e r
c o n t r o l l e d c o n d i t i o n s . I t c o n s i s t s o f a b o t t o m l e s s b o t t l e ( 7 0 0 m l c a p a c i t y ) ,
w i t h a w i c k f i x e d i n t h e h e c k , i n v e r t e d i n t o a l i t t e p r e s e r v i n g j a r C O l l t a i n i n g
q u a r t e r s t r e n g t h n u t r i e n t s o l u t i o n 7 . T h e b o t t l e i s n o r m a l l y f i l l e d w i t h s a n d
a n d t h e w h o l e u n i t a u t o c l a v e d p r i o r t o s o w i n g . T h e U l l i t w a s u s e d i n t h e s e
s t u d i e s as a n a l t e r n a t i v e t o t u b e s i n t h e h o p e t h a t i t wo u l d b e pos s i bl e t o
wo r k wi t h a l a r g e r a mo u n t of sol l ( up t o 200 g) a n d so p e r mi t d e t e c t i o n of
r h i z o b i a o c c u r r i n g as a v e r y s p a r s e p o p u l a t i o n . A c o r r e s p o n d i n g q u a n t i t y of
s a n d h a d , of cour s e, t o b e r e mo v e d f r o m t h e b o t t l e j a r t o a c c o mmo d a t e t h e
l a r ge a mo u n t of sol l .
(b) P l a n t t u b e s . Te s t t u b e s (15 c m 2 cm) , c o n t a i n i n g 8 - 1 0 ml of 0 . 8 %
a g a r me d i u m 7 we r e p h g g e d wi t h c o t t o n wool a n d s t e r i l i s e d. Se e d wa s s o wn
e s s e n t i a l l y as a l r e a d y d e s c r i b e d 2. Ag a r t u b e s we r e g e n e r a l l y s l o p e d f or l a r ge
a l i quot s , b u t a g a r de e ps ( " p l u g s " ) we r e s i mpl e r , a n d y i e l d e d e q u a ] l y a e c u r a t e
r e s u l t s wi t h v o l u me s u p t o 0. 5 r el . S a n d r e p l a c e d t h e a g a r i n s o me c o mp a r a t i v e
t r i a l s .
Al l e s t i ma t e s of t h e n u mb e r of r h i z o b i a we r e b a s e d o n t h e a p p r o p r i -
a t e t a b l e s of F i s h e r a n d Ya t e s 2 3. Th e s e c a l c u l a t i o n s a r e s i mp l e a n d
f l e xi bl e ( a c c or di ng t o n a t u r e a n d n u mb e r of d i l u t i o n s t e p s a n d n u mb e r of
r e p] i c a t e s ) a n d a l l ow f i duc i a l l i mi t s t o b e r e a d i l y d e t e r mi n e d . *
Qualitative, direct, methods
(a) F i e l d s o wi n g s . Th e s i mp l e s t me t h o d of d e t e c t i n g r h i z o b i a i n sol l i s t o
l o o k f or n o d u l e s o n p l a n t s r a i s e d f r o m s ur f a e e - s t e r i l i s e d s e e d s own d i r e c t l y
i n t o t h e sei l , t r e a t e d as a p p r o p r i a t e t o p r o v i d e good c o n d i t i o n s f or n o d u l a t i o n .
P r o v i d e d t h e r o o t s y s t e ms of a d j o i n i n g p l a n t s a r e n o t i n t e r - mi n g l e d , t h e
r e s u l t s can, f or c o mp a r a t i v e p u r p o s e s , b e e x p r e s s e d as p e r c e n t a g e of t h e
p l a n t s t h a t h a v e f o r me d nodul e s . I t i s n o t pos s i bl e t o pl a c e a n y g r e a t q u a n t i -
t a t i v e e mp h a s i s o n t h e r e s ul t , h u t i t c a n gi ve us e f ul i n f o r ma t i o n as t o t h e
d i s t r i b u t i o n a n d s p r e a d of r h i z o b i a , a n d c a n b e u s e d t o p r e d i c t wh a t wo u l d
h a p p e n i n a s o wi n g of u n i n o c u l a t e d s e e d i n t h e s a me a r e a .
(b) S o l l c o r e a s s e mb l y . Th e s a me p r i n c i p l e i s f ol l owed, wi t h t h e n n i t of
s t u d y b e t t e r de f i ne d, wh e n c a n s (12 7. 5 cm) s e r v i n g as o p e n - e n d e d c yl i n-
der s , a r e d r i v e n i n t o t h e g r o u n d t o enel os e cor es of soil. Th e e ne l os e d sol l c a n
t h e n b e ' e x p l o r e d ' f or r h i z o b i a b y s o wi n g a n u mb e r of s ur f a e e - s t e r i l i s e d s e e ds
* The t er m ' most probabl e nurnber (MPN)' as used i n t he work report ed i n t he
present paper rel at es {o t he approxi mat e est i mat e based on t abl e VIII2 of Fi s h e r
and Ya t e s .
DE T E C T I ON AND E S T I MAT I ON OF RHI Z OBI A I N SOI L 7 5
i n e a c h c o r e . C r o s s - c o n t a mi n a t i o n h a s , o f c o u r s e , t o b e a v o i d e d a n d c o n d i t i o n s
ma d e s a t i s f a c t o r y f o r n o d u l e f o r ma t i o n .
T h e c h o i c e wh e t h e r t o r e mo v e t h e e n c l o s e d c o r e o f s oi l ( wi t h a s e p t i c p r e -
c a u t i o n s ) o r l e a v e i t i n situ d e p e n d s o n c o n d i t i o n s ( r a i n f a l l , t e mp e r a t u r e ) .
I n a n y c a s e t h e s p a t i a l r e l a t i o n s h i p o f t h e t o p 12 c m o f s o i l c a n b e r e t a i n e d
d u r i n g t h e c o u r s e o f t h e t e s t . I n t h e s e s t u d i e s c o r e s we r e t r a n s p o r t e d i n p l a s t i c
j a r s , wh i c h p r o v i d e d p r o t e c t i o n f r o m c o n t a mi n a t i o n a n d a l l o we d wa t e r i n g
f r o m t h e b o t t o m o f t h e c o r e d u r i n g t h e c o u r s e o f t h e e x p e r i me n t .
T h e p r e s e n c e o r a b s e n c e o f n o d u l e s i n t h i s c a s e c a n b e e x p r e s s e d a s a t l e a s t
o n e ( or l e s s t h a n o n e ) r h i z o b i u m p e r s oi l c o r e ( a p p r o x . 7 0 0 g) .
RE S UL T S
A. Recovery o/ rhi zobi a/ rom a counted p@ul at i on
Co mp a r i s o n s of a s s e mb l y a n d c o n d i t i o n s of t e s t i n g . The
quant i t at i ve r ecover y of rhi zobi a in pure cul t ure (checked agai nst
t he regul ar pl at e count) was compar ed wi t h several assemblies and
t wo condi t i ons of t est i ng (Table 1). Agar t ubes gar e sat i sf act or y
r ecover y (except on occasion 3 in t he glasshouse where t emper at ur e
had been excessive), and were superi or to t he ot her assemblies.
T ABL E 1
I nf l uence of a s s e mb l y a nd gr owt h condi t i ons oll r e c ove r y of r hi zobi a f r om c ount e d
pur e s us pe ns i ons
Pl at e e s t i ma t e s Pl a n t t e s t s
Oc-
casi on
Log pI at e
col ony Log pl at e
MPN
c o u n t
2. 64 2.19
1. 75 1. 77
9. 32 10.00
Condi t i ons of gr owt h
As s e mb l y Locat i on
Agar t ube s Li ght r oom
Agar t ube s Li ght r o o m
Agar t ube s Li ght r o o m
Agar t u b e s Gl as s hous e
Sa n d t ube s Li ght r o o m
Sa n d t ube s Gl as s hous e
Sa n d bot t l es Gl as s hous e
Log p l a n t
MPN
2. 52
1. 77
9.49
8.49
7.24
8.00
7. 76
Log pl a t e
coi ony
c o u n t - - l o g
p l a n t MPN
0. 12
- - 0. 02
- - 0. 17
0.83
2.08
1. 32
1. 56
95% f i duci al l i mi t s for l og MPN e s t i ma t e s on occasi on 3 = - - 0.54.
De t a i l e d p r o c e d u r e s wi t h a g a r t u b e s . Tabl e 2 gives results
of six compari sons bet ween t he pl at e count and pl ant count s wi t h
each of six t est i ng procedures. All showed a difference in f avour of
t he pl at e count , but general l y t he pl ant count was as good as t he
pl at e-MPN. Tr eat ment 2 was t he procedure nor mal l y followed in
t hese studies.
76 j . A. THOMPSON AND J. M. VI NCENT
TABLE 2
Effect of modi fi cat i ons of agar-t ube t echni que on recovery of rhi zobi a from a
count ed pure suspension
Procedure Log pl ant
MPN
Tr eat ment
No.
Seed
Sterilised
Sterilised
Sterilised
Sterilised
Unsterile
Unsterile
Sowing
I mmedi at e
I mmedi at e
Soaked
Soaked
I mmedi at e
I mmedi at e
I Inocul at i on
I mmedi at e
Del ayed
I mmedi at e
Del ayed
I mmedi at e
Del ayed
1. 67
2.11
1. 98
1. 79
1. 98
1.98
Log pl at e count --
Log pl ant MPN
0.52
0.08
0.21
Q.40
0.21
0.21
Log pl at e count = 2.19 Log pl at e MPN = 1.87
95% fiducial l i mi t s for log MPN wi t h each t r eat ment = ~ 0.36
B. Recovery o~ rhizobia /rom soil
Th e e f f e c t of a s s e mb l y . Pr evi ous i ndi cat i ons t hat t he agar
t ube mi ght be unf avour abl e t o t he r ecover y of rhi zobi a from sparse
popul at i ons in t he soil, l ed us t o t est t he l arger assembl y of t he bot t l e
j ar, and t o subst i t ut e sand for agar in t he t ube. Al t hough experi ence
wi t h count ed suspensi ons of rhi zobi a in pure cul t ur e was inferior
wi t h t hese modi fi cat i ons (Table 1), t he use of sand di d in Iact show
a t r end (not st at i st i cal l y significant) t owar ds bet t er r ecover y from
soil (Table 3).
TABLE 3
The effect of assembl y in t he det ect i on of rhi zobi a in soff*
Number of assemblies
Form of assembl y
Wi t h nodules Wi t hout nodul es
Agar t ubes . . . . . . . . . . . 16 32
Sand tubes . . . . . . . . . . . 23 25
Bot t l e jars (sand) . . . . . . . . . 24 24
* Added in equal number of 1-g and 0.1-g sampl es for each assembly.
X~, agar (tubes) r s . , sand (tubes and bottles) 3. 2, P < 0.10 and > 0.05.
Co mp a r i s o n of i n d i r e c t Co u n t s a n d d i r e c t n o d u l a t i o n
t r i al s. Twent y- f our field pl ot s were exami ned for survi vi ng cl over
rhi zobi a as j udged b y t hei r abi l i t y t o nodul at e sown sterilised seed
and i ndi rect l y b y means of t he pl ant di l ut i on count . The di rect
met hods i nvol ved:
(i) sowing a short row of seed in each pl ot ; and
DETECTI ON AND ESTI MATI ON OF RHI ZOBI A I N SOI L 7 7
(ii) removing four soil cores from each plot, sowing t hem with
sterilised seed and maintaining t hem in the glasshouse.
The count was done on a 2 cm wide 5 cm deep sample, taken
from each core, mixed thoroughly and used for an MPN estimate by
the agar tube method.
The results are summarised in Table 4, grouping the samples ac-
cording to the indirect quant i t at i ve estimate. The two direct
methods agree well (r = 0.77, P < 0.01; based on arc sin ~/per
cent). Wi t h one exception, the seedlings, whether sown in field or
core, were all nodulated when the recoverable rhizobia exceeded
1 per g of soil. All cases but one of less t han 1 rhizobium per gram
were accompanied by reduced nodulation in bot h the field (mean,
55 per cent) and core methods (65 per cent).
TABLE 4
Di r ect and i ndi r ect de mons t r a t i on of r hi zobi a i n soll
% Nodul at ed Pl a nt s Soils gr aded b y pl a nt di l ut i on count
(per g soil)
> 10
1-10
< 1
479
38
36
10
5
5
2
Fi el d [ Core
100 / 100 /
1~60/ 95 170l 92
100 ] 100 "
/
100 [ 100
1 0 0 / 100 100 ' 100.
1 0 o j 1 0 0 ,
78 50
70 63
58 . 55 100~ 65
J
53 63
14 50
The degree of nodulation in the sown soll was more t han one might
have expected from the counts. The roots of the plants from field
t reat ment s with less t han 100 per cent nodulation carried few nod-
ules at the top 5-cm zone from which the count had beep made.
Nodulation of the plants sown in field or core would t hen have
reflected an exploration of a greater depth of soff.
Re c ove r y at l ower soi l di l ut i ons . A large collection of
samples from what appeared to be a sparsely populated soil was tested
78 j . A. T HOMP S ON AND J . M. VI NC E NT
for t he nodul at i on of cl over wi t h t he agar t ube met hod, using four in-
creasing di l ut i ons equi val ent t o 1, 0.1, 0.01, and 0.001 g soil per t ube.
Si xt y-one sampl es gave at l east one posi t i ve t ube but some of t hese
t hough posi t i ve at higher dilution were negat i ve at a lower di l ut i on
(more soil). I t was essential t o arri ve at an apprai sal of t he signifi-
cance of such skips.
To faci l i t at e present at i on, t he negat i ve t ubes have been classified
as follows and i l l ust rat ed wi t h t wo r epr esent at i ve sampl es (Table
5A): Sk@ - negat i ve t ubes at l east one t enfol d di l ut i on bel ow a
posi t i ve t ube (A1) ; t wo t en-fol d dilutions bel ow (A2) ; Paired negative
- negat i ve t ube at t he same di l ut i on as t he hi ghest posi t i ve t ube
(B); Normal negative - negat i ve t ube at one t en-fol d di l ut i on above
t he hi ghest posi t i ve (C) or at t he l owest di l ut i on of a whol l y negat i ve
series (D).
Obser vat i ons of negat i ve t ubes showed t hat t he seedlings carri ed
r oot hai rs so t hat i nfect i on sites were available. Tubes were t hen
exami ned for t he presence of rhi zobi a b y breaki ng up t he agar and
t he pl ant , and t hor oughl y shaki ng wi t h glass beads and 10 ml sterile
T AB L E 5
Re - t e s t of c o n t e a t s of p o s i t i v e a n d n e g a t i v e p l a n t t n b e s
A. S a mp l e d e s i g n a t i o n of n e g a t i v e t u b e s
We i g h t of s oi l i n s u b - s a mp l e (g)
S a mp l e R e p l i c a t i o n
1 I 0 . 1 [ O . O 1 I 0. 001
1 ( i ) + + - - ( B ) - - ( C )
(ii) - - (A~) i - - (A1) + - - (C)
i
i
2 (i) - - ( D ) ,] - - - - - -
( i i ) - - ( D ) [ - - - - - -
B. Re - t e s t r e s u l t *
S t a t u s a t I s t t e s t No d u l a t i n g [ No n - n o d u l a t i n g P e r e e n t +
P o s i t i v e .
S k i p s A~
S k i p s A1
P a i r e d n e g a t i v e s B
No r ma l n e g a t i v e s C
No r ma l n e g a t i v e s D . . . .
22
9
12
9
5
4
1
6
15
3O
46
114
96
60
45
23
10
3
* + t u b e i n t h e r e - t e s t i n d i c a t e s t h e p r e s e n c e of a t l e a s t 2 0 r h i z o b i a i n t h e s a mp l e d t u b e .
DE T E C T I ON AND E S T I MAT I ON OF R HI Z OB I A I N S OI L 7 9
water. Duplicate 0.5-ml aliquots were t hen added to freshly grown
plants in agar tubes and later examined for nodulation. Results
along with a check of positive tubes are given in Table 5B.
Few of the normal negatives were re-graded positive (C > D).
Skips were commonly positive (A' > A,) and paired negatives were
intermediate between normal negatives and skips. The failure of
some skips to yield rhizobia might reflect a very uneven distribution
of rhizobia between aliquots, but could as likely be due to failure of
the few rhizobia to survive in a competitive, and perhaps antago-
nistic, biotie environment in the original tube.
C. In]luence o/added soil on the recovery o/rhizobia
Complete evaluation of the dat a obtained above was prevented
by the lack of any preeise information on the actual number of
rhizobia. A comprehensive experiment was therefore designed to
examine the effect of soil added to the tube on detection of known
numbers from a pure suspension. The results with 8 replicates of a
4-fold serial dilution are shown in Table 6.
T AB L E 6
R e e o v e r y of r h i z o b i a f r o m p u r e c u l t u r e wi t h a n d wi t h o u t a d d i t i o n of s oi l t o a g a r t u b e s
Ad d e d s ol l ( g / t u b e )
S t e r i l i s e d ] Un s t e r i l e
0 0
0 0. 01
0 1
1 0
1 0 . 0 1
1 1
T u b e s wi t h n o d u l e s f o r
S u c c e s s i v e F o u r f o l d Di l u t i o n s *
L o g MP N
3 2. 52
3 2. 71
0 2. 03
4 2. 91
0 2. 19
0 1. 49
E x p e c t e d P o s i t i v e s
P o s i t i v e P l a t e s
C a l c n l a t e d r h i z o b i a
p e r t u b e * * 4 0 0 100 25
9 5 % f i d u e i a l l i mi t s f o r l o g MP N of e a e h t r e a t me n t
* 8 t u b e s t e s t e d a t e a e h d i l u t i o n .
** B a s e d o n p l a t e s e o u n t e d a t s u i t a b l e d i l u t i o n s .
8 8
8 3
6 1. 6
= -I-0.33
3 2. 61
1 2 . 1 9
0. 4
Detection of very small numbers was poor in the presence of 1 g
soil, whether unsterile or re-contaminated with some unsterile soil.
In fact, the presence of a large amount of unsterile soll could 1essen
8 0 J . A. T HOMP S ON AND J . M. VI NC E NT
rel i abi l i t y er en wi t h t he addi t i on of a fai rl y large number of rhizobia.
Two f ur t her exper i ment s gar e small, t hough not significant, de-
pressions when 1 g soll was added to agar pl ant - t ubes used in t he
r ecover y of count ed rhizobia.
Tabl e 7 gives results of a mor e comprehensi ve exper i ment ex-
ami ni ng r ecover y of (A) a count ed pure cul t ure and (B) a di fferent
popul at i on in soil itself. The addi t i on of unst eri l e soff caused a con-
si st ent depression whi ch was mor e mar ked when cont ami nat i ng soil
mi cro-organi sms had had addi t i onal t i me to become est abl i shed be-
lore i nocul at i on (A iii). The addi t i onal soff represent ed in (B iv) ap-
pears t o have exercised a f ur t her depressing effect.
T ABL E 7
Re c o v e r y of r hi z obi a a f t e r t he a ddi t i on of uns t e r i l e soff t o a ga r t u b e s
Soff a dde d at Soff a dde d 7 da ys
Rhi zobi al s our e e i noc ul a t i on be f or e i noc ul a t i on Log MPN*
(g) (g)
A.
B.
(i) Pur e c ul t ur e
(ii) Pur e c ul t ur e
(iii) Pur e c ul t ur e
(iv) Pur e c ul t ur e
(i) Soi l s u s p e n s i o n
(ii) Soil s us pe ns i on
(iii) Soi l s us pe ns i on
(iv) Soff s us pe ns i on
(v) Sol l -i n-sol l
(vi) Soi l -i l l -soi l
q
0. 5
0. 5
m
0. 5
0.5
0.5
0.5
0.5
0.5
0.5
0. 5
0.5
1. 77
1. 37
0. 88
0. 88
> 4. 26
3. 92
3. 68
2. 99
2.13
1. 77
* Le a s t s i gni f i cant di f f er ence ( P = .0S), 0.65.
An at t empt to si mul at e t he condi t i on of havi ng few rhizobia in
t he soll, and havi ng t he t ot al soll recei ved by each t ube const ant ,
was made di l ut i ng t he soil being assayed in rhi zobi um-free soff (suc-
cessively by first breaki ng t he aggregat es t hor oughl y in mor t ar and
pestle, and t hen mi xi ng in t hor oughl y wi t hout addi t i on of wat er
(Table 7, B v, vi) ). The r ecover y was ver y poor when compar ed wi t h
t he corresponi ng wat er - di l ut ed series (B iii and iv). This poor re-
cover y seemed first t o i ndi cat e t hat t here had been i nadequat e dis-
persion of t he initial sampl e in successive dilutions. An i ndependent
check done wi t h t he 10 and 100 soil-in-soil dilutions showed
reasonable agr eement bet ween t hem (900 a n d 150 recovered). Ap-
DE T E C T I ON AND E S T I MAT I ON OF R HI Z OB I A I N S OI L 81
plying these results, the relationship between number of rhizobia
added per tube can be set out as follows (using an average figure for
the best estimate)"
Amo u n t of soi l
I l g I
Ex p e c t e d f r o m B (i) ( Tabl e 7) . . 18000
I n t e r n a l check 12000
Ex pe c t e d + t u b e s . 8
, F o u n d + t u b e s . . . . . . . . I 8 ,
0 . l g
1800
1200
8
8
0lg ] . 00l g
180 18
120 12
8 8
6 1
[ . 0 0 0 l g
1.8
1.2
6
0
It seemed likely, therefore, t hat rhizobia were present but un-
detected at the greater dilutions. This possibility was confirmed
when the tube contents of the only ' s k i p ' , and of Iour ' paired nega-
tives' were resuspended in water and tested for the presence of rhi-
zobia as already described. All were positive on re-test as was one of
two "normal negatives" at dilution 5. Failure to suspend the rhi-
zobia in liquid suspension, had interfered with detection, probably
because of greater difficulty in establishing contact with the root
surface.
DI S C US S I ON
The present dat a afford further evidence of the reliability of a
plant-dilution count of pure suspension of clover rhizobia when
based on agar slopes or deeps. They can be summarised in terms of 11
comparisons with the plate counts and the lant-dilution count t hat
yielded the following average Iigures:
Log pl a t e c o u n t I Log pl a t e MPN Log p l a n t MP N
4. 18 ] 4.09 4. 17
The presence of soil, however, can seriously affect the quantitative
recovery of rhizobia. The difficulty was shown in the first instance
when soils having rhizobia sufficient to permit nodulation at higher
dilutions had excessive negative ( " s k i p " ) tubes at lower dilutions in
the presenee of a relatively large amount of unsterile soil. The fact
t hat a large proportion of these skip tubes (45 to 6o per cent - Table
5B) was found to contain viable clover rhizobia on further exami-
nation argued against failure to disperse the bacteria randomly, and
indicated rather t hat a sufIiciently unfavourable environment had
82 J . A . T H O M P S O N A N D J . M . V I N C E N T
been est abl i shed in t he ski p t ube t o pr event nodul at i on. I t is also
qui t e l i kel y t hat some of t he ski p t ubes, in whi ch rhi zobi a coul d not
be demonst r at ed b y f ur t her isolation, had become suffi ci ent l y un-
f avour abl e for t hei r inhibition or deat h.
Posi t i ve evi dence of t he unf avour abl e act i on of a r el at i vel y large
amount of unst eri l e soil is pr ovi ded in Tabl es 6 and 7. The i nt erferi ng
effect was a funct i on of amount of soil, its cont ent of ot her micro-
organi sms (a r el at i vel y small qnant i t y of unst eri l e soil (.01 g) bei ng
abl e t o creat e an unsat i sf act or y t ube envi r onment wi t h 1 g sterile
soil), and t he l engt h of t i me t he i nt erferi ng popul at i on had t o es-
t abl i sh itself (Table 7).
The di ffi cul t y in t he case of soils havi ng at l east a few hundr ed
rhizobia per g is not l i kel y t o be t oo serious, in t hat higher di l ut i ons
are l i kel y t o yi el d posi t i ves and j udi ci ous not i ce can be t aken of
skips, ei t her quant i t at i vel y or in present i ng and i nt erpret i ng t he
results. The real pr obl em lies in t he val i di t y of det er mi nat i ons wi t h
ver y l ow number s for whi ch t he dilutions expect ed t o be posi t i ve
woul d have t he combi nat i on of a large amonnt of soil (1-0.1 g) and
few rhizobia.
The first appr oach t o this is t o subst i t ut e for t he agar t ube an
assembl y whi ch might, because of a more porous suppor t mat er i al
and/ or its size, per mi t larger sampl es of soil t o be added wi t hout
havi ng t he same undesi rabl e effects. In fact t he t r end t owar ds su-
per i or i t y of bot t l e jars, and even sand t ubes, over agar t ubes, in
Tabl e 3 wonl d be encouragi ng were it not for t hei r consi derabl e
i nferi ori t y in recoveri ng rhi zobi a in pure cul t ur e t est s (Table 1).
Br o c k we l l 1 also f ound vermi cul i t e t ubes under est i mat ed a pure
cl over popul at i on.
The experi ment i n whi ch l ow-count soils were si mul at ed by soil-
in-soil dilution (Table 7B, v and vi) not onl y emphasi sed t he diffi-
cul t y of recoveri ng a few rhi zobi a in a r el at i vel y large amount of soll
but also accent uat ed a possible spat i al fact or t hat coul d del ay con-
t act bet ween t he few rhizobia ( pr obabl y in discrete micro-colonies)
and t he r oot surface. The addi t i on of sufficient wat er wi t h t he soil
and some degree of agi t at i on in t he t ube woul d pr obabl y over come
this par t of t he probl em.
Fi nal l y our experience emphasi ses t he val ue of direct, t hough onl y
semi - quant i t at i ve, met hods for t est i ng for rhizobia in sparsel y popu-
l at ed solls. Soil amelioration, f r eedom from cont ami nat i on and
DETECTI ON AND ESTI MATI ON OF RHI ZOBI A IN SOIL 83
maintenance of adequate moisture will all be i mport ant in such tests.
The fact t hat the rhizobium-free seedling will be able to explore a
reasonable volume of soll means t hat it can provide information on
the spatial di st dbut i on of rhizobia and give some idea of their rela-
tive abundance.
The search for better assemblies and substrates needs to be per-
sisted with in order to fill the gap in the quant i t at i ve assessment of
the sparsely populated soil. Meanwhile a combination of the plant
dilution count in agar tubes and direct sowing of rhizobia-free seed
can be depended onto define the situation within reasonable limits.
Some degree of secondary inoculation, looking for rhizobia in se-
lected negative tubes, could help with the plant-dilution count in
the more i mport ant difficult cases (comp. Table 5B).
SUMMARY
Th e p l a n t - i n f e c t i o n t e c h n i q u e f or t h e e s t i ma t i o n of r hi zobi a, i n whi c h
s ma l l - s e e de d hos t s a r e gr own on a ga r wi t h i n t e s t - t ube s , is a ppl i c a bl e t o soi l s
wi t h a mo d e r a t e r hi z obi a l p o p u l a t i o n (in t h e or de r of a t l eas t 100/g). Ac c o u n t
mi g h t h a v e t o be t a k e n of "skips" (less d i l u t e d : ne ga t i ve , wh e n mor e di l ut e d
a r e pos i t i ve) l i ke l y t o r es ul t , a t l e a s t i n pa r t , I r om u n f a v o u r a b l e c ondi t i ons f or
r hi z obi a l s ur vi va l , mu l t i p l i c a t i o n or nodul a t i on. Be c a us e of s uch ef f ect s, a
s pa r s e p o p u l a t i o n (in t h e or de r of (10/g) ma y n o t be d e t e c t e d e ve n wi t h o u t
di l ut i on (1 g soi l pe r p l a n t t ube) . Loc a l i s a t i on of r hi z obi a i n t h e soi l is l i kel y
t o be i mp o r t a n t i n d e t e r mi n i n g c o n t a c t wi t h t h e p l a n t r oot s i n t h e di l ut i on
c o u n t a n d i n s a mp l i n g f r om t h e fi el d. Di f f i cul t i es wi t h s pa r s e l y p o p u l a t e d
soi l s c a n be p a r t l y o v e r c o me b y c a r e f ul l y c o n d u c t e d di r e c t sowi ngs of st er i l i sed
seed, p r e f e r a b l y i n t h e COllIines of cores, e i t he r l e i t i n t h e f i el d or b r o u g h t b a c k
t o t h e gl as s bous e.
ACKNOWLEDGMENT
Th e wo r k r e p o r t e d i n t hi s p a p e r has been s u p p o r t e d b y r e s e a r c h f unds
f r o m t h e Un i v e r s i t y of Sydne y, t h e Ru r a l Cr e di t s De v e l o p me n t F u n d of t h e
Re s e r v e Ba n k of Au s t r a l i a a nd b y Wh e a t I n d u s t r y Re s e a r e h Funds .
Received October 25, 1965
84 DETECTI ON AND ESTIMATION OF RHIZOBIA IN SOIL
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