rhyzobium

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P l a n t a n d S o i l X X V I , n o . 1 F e b r u a r y 1 9 6 7

M E T H O D S O F D E T E C T I O N A N D E S T I M A T I O N O F

R H I Z O B I A I N S O I L

b y J . A . T H O M P S O N * a n d J . M . V I N C E N T * *

M i c r o b i o l og y L a b o r a t o r y , S c h o o l f A g r i c u l t u r e , U n i v e r s i t y o f S y d r l ey ,

S y d n e y , N e w S o u t h W a l e s , A u s t r a l i a

I N T R O D U C T I O N

D e t e c t i o n o f s p e c if ic r h i z o b i a i n so ll , a n d t h e i r q u a n t i t a t i v e d e -

t e r m i n a t i o n , a r e e s s e n t i a l f o r e c o l o g i c a l s t u d i e s a i m e d a t d e f i n i n g

t h e f a t e o f r h i z o b i a o n s e e d a n d s e e d l in g , a n d t h e i r p e r s i s t e n c e f o r

l o n g e r p e r i o d s i n t h e s o l l a n d r h i z o s p h e r e . S u c h d e f i n i t i v e i s o l a t i o n

o f r h i z o b i a f r o m t h e s o i l , i n w h i c h a t t h e b e s t t h e y a r e l i k e l y t o

c o n s t i t u t e a s m a ll a n d n o t v e r y v i g o r o u s p a r t o f t h e t o t a l p o p u l a t i o n ,

r e s t s o n t h e i r c a p a c i t y t o n o d u l a t e a s p e c if ic l e g u m i n o u s h o s t . P r e -

c is i on i n d e t e r m i n i n g t h e n u m b e r o f r h i z o b i a w il l t h e n d e p e n d o n t h e

a g r e e m e n t b e t w e e n t h e n u m b e r i n t h e so il a n d t h e n u m b e r e s t i m a t e d

b y w h a t i s n e c e s s a r i l y a n i n d i re c t m e t h o d . T h e p r i n c ip l e o f t h i s

i n d i r e c t ( p l a n t - d i l u t i o n o r p l a n t i n f e c t i o n ) c o u n t ( W i l s o n 13)

h a s b e e n f o ll o w e d b y o th e r s i n t e r e s te d i n t h e q u a n t i t a t i v e r e c o v e r y

o f rh i z o b i a f r o m s o il a n d o t h e r b i o l o g i c a l ly c o m p l e x s i t u -

a t i o n s 1 2 6 10 11. P u r e c u l t u r e s o f rh i z o b i a a p p e a r t o o f f e r n o p a r t i -

c u l a r d i f f i c u l t y i n o b t a i n i n g r e l i a b l e c o u n t s b y a p l a n t - d i l u t i o n

t e c h n i q u e i n tu b e s . A c o n s i d e r a b l e b o d y o f p u b l i s h e d 2 a n d u n -

p u b l i s h e d d a t a f r o m t h i s l a b o r a t o r y s h o w e d l it t le d i f f er e n c e b e -

t w e e n t h e p l a t e a n d p l a n t c o u n t s f o r m e a n s b a s e d o n 5 0 c o m p a r is o n s

w i t h c l o v e r a n d 3 4 w i t h l u c e r n e .

T h e s i t u a t i o n a s t o r e c o v e r y f r o m s o il i s l e s s c l e ar . I t i s d i f f i c u l t t o

i n t e r p r e t T u z i m u r a a n d W a t a n a b e ' s f i n d i n g s i n t h e p r e s en c e o f s o il

P r e s e n t a d d r e s s : N e w S o u t h W a l e s D e p a r t m e n t o f A g r i c u l t u re , R e s e a r c h S t a t i o n ,

T a m w o r t h , N . S . W . , A u s t r a l i a .

P r e se l xt a d d r e s s : S c h o o l o f B io l o g ic a l S c ie n c e s , U l l i v e r s i t y o f N e w S o u t h W a l e s ,

K e n s i rl g t or l , N . S . W . , A u s t r a l i a .

7 2

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D E T E C T I O N A N D E S T I M A T I O N O F R H I Z O B I A I N S O IL 7 5

i n e a c h c o r e . C r o s s - c o n t a m i 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

m a 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 m a t i o n .

T h e c h o i c e w h e t h e r t o r e m o v e t h e e n c l o s e d c o r e o f s o i l ( w i 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 n s tu 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 m p 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 1 2 c m o f s o il 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 w e 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 , w h 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 m i n a t i o n a n d a l l o w e d w a 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 m e 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 ( o r l e s s t h a n o n e ) r h i z o b i u m p e r s o i l c o r e ( a p p r o x . 7 0 0 g ) .

R E S U L T S

A R ec ov er y o / r h i z o b i a / r o m a c ou nte d p @ u l a t i o n

C o m p a r i s o n s o f a s s e m b l y a n d c o n d i t i o n s o f t e s t i n g . T h e

q u a n t i t a t i v e r e c o v e r y o f r h i z o b ia in p u r e c u l t u r e c h e c k e d a g a i n s t

t h e r e g u l a r p l a te c o u n t ) w a s c o m p a r e d w i t h s e v e r a l a s se m b l ie s a n d

t w o c o n d i t i o n s o f t e s t i n g T a b l e 1). A g a r tu b e s g a r e s a t i s f a c t o r y

r e c o v e r y e x c e p t o n o c c as io n 3 i n th e g l a ss h o u s e w h e r e t e m p e r a t u r e

h a d b e e n e x c e s s iv e ) , a n d w e r e s u p e r i o r t o t h e o t h e r a s s e m b li e s.

T A B L E 1

I n f l u e n c e of a s s e m b l y a n d g r o w t h c o n d i t io n s o ll re c o v e r y o f r h i z o b i a f r o m c o u n t e d

p u r e s u s p e n s i o n s

P l a t e e s t i m a t e s P l a n t t e s t s

O c -

c a s i o n

L o g p I a t e

c o l o n y L o g p l a t e

M P N

c o u n t

2 . 6 4 2 . 1 9

1 7 5 1 7 7

9 . 3 2 1 0 . 0 0

C o n d i t i o n s o f g r o w t h

A s s e m b l y L o c a t i o n

A g a r t u b e s L i g h t r o o m



A g a r t u b e s G l a s s h o u s e

S a n d t u b e s L i g h t r o o m

S a n d t u b e s G l a s s h o u s e

S a n d b o t t l e s G l a s s h o u s e

L o g p l a n t

M P N

2 . 5 2

1 7 7

9 . 4 9

8 . 4 9

7 . 2 4

8 . 0 0

7 . 7 6

L o g p l a t e

c o i o n y

c o u n t - - l o g

p l a n t M P N

0 . 1 2

- - 0 . 0 2

- - 0 . 1 7

0 . 8 3

2 . 0 8

1 3 2

1 5 6

9 5 f i d u c i a l l i m i t s f o r l o g M P N e s t i m a t e s o n o c c a s i o n 3 = - - 0. 54 .

D e t a i l e d p r o c e d u r e s w i t h a g a r t u b e s . T a b le 2 g iv es r es u l t s

o f s ix c o m p a r i s o n s b e t w e e n t h e p l a t e c o u n t a n d p l a n t c o u n t s w i t h

e a c h o f s ix t e s t i n g p r o c e d u r e s . A l l s h o w e d a d i f f e r e n c e i n f a v o u r o f

t h e p l a t e c o u n t , b u t g e n e r a l l y t h e p l a n t c o u n t w a s a s g o o d a s t h e

p l a t e - M P N . T r e a t m e n t 2 w a s t h e p r o c e d u r e n o r m a l l y f o ll o w e d i n

t h e s e s t u d i e s .

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7 6 j . A .

THOMPSON AND J. M. VINCENT

TABLE 2

Effect of modificati ons of agar- tube techniq ue on recover y of rhizobi a from a

counted pure suspension

Procedure Log plant

MPN

Treatment

No.

Seed

Sterilised

Sterilised

Sterilised

Sterilised

Unsterile

Unsterile

Sowing

Immedia te

Immediate

Soaked

Soaked

Immediate

Immediate

I Inoculation

Immediate

Delayed

Immediate

Delayed

Immediate

Delayed

1 6 7

2.11

1 9 8

1 7 9

1 9 8

1 9 8

Log plate count --

Log plant MPN

0.52

0 08

0.21

Q 40

0.21

0.21

Log pla te coun t = 2.19 Log pl ate MPN = 1.87

95 fiducial limi ts for log MPN with each tr eat men t = ~ 0.36

B Recovery o~ rhizobia /ro m soil

T h e e f f e c t o f a s s e m b l y . P r ev i o us i nd ic a t io n s th a t t h e a g ar

t u b e m i g h t b e u n f a v o u r a b l e t o t h e r e c o v e r y o f r h i z o b ia f r o m s p a rs e

p o p u l a t i o n s i n t h e s o il , l e d u s t o t e s t t h e l a r g e r a s s e m b l y o f t h e b o t t l e

j a r , a n d t o s u b s t i t u t e s a n d f o r a g a r i n t h e t u b e . A l t h o u g h e x p e r i e n c e

w i t h c o u n t e d s u s p e n s i o n s o f r h i z o b i a i n p u r e c u l t u r e w a s i n f e ri o r

w i t h t h e s e m o d i f i c a t i o n s T a b l e 1), t h e u s e o f s a n d d i d in I a c t s h o w

a t r e n d n o t s t a t i s t i c a l l y s i g n if ic a n t) t o w a r d s b e t t e r r e c o v e r y f r o m

so i l Ta b le 3 ) .

TABLE 3

The effect of ass embl y in the detect ion of rhizobia in soff*

Number of assemblies

Form of assembly

With nodules With out nodules

Agar tubes . . . . . . . . . . . 16 32

Sand tubes . . . . . . . . . . . 23 25

Bottle jars (sand) . . . . . . . . . 24 24

* Added in equal number of 1-g and 0.1-g samples for each assembly.

X~ agar (tubes) r s . sand (tubes and bottles) 3 . 2 P < 0.10 and > 0.05.

C o m p a r i s o n o f i n d i r e c t C o 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 a l s . T w e n t y - f o u r f i e ld p l o t s w e r e e x a m i n e d f o r s u r v i v i n g c l o v e r

r h i z o b i a a s j u d g e d b y t h e i r a b i l i t y t o n o d u l a t e s o w n s te r il i s ed s e e d

a n d i n d i r e c t l y b y m e a n s o f t h e p l a n t d i lu t io n c o u n t . T h e d i r ec t

m e t h o d s i n v o l v e d :

i) s o w i n g a s h o r t r o w o f s e e d i n e a c h p l o t ; a n d

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D E T E C T I O N A N D E S T I M A T I O N O F R H I Z O B I A I N S O I L 7 7

ii) removing four soil cores from each plot, sowing them with

sterilised seed and maintaining them in the glasshouse.

The count was done on a 2 cm wide 5 cm deep sample, t aken

from each core, mixed thoroug hly and used for an MPN estimate b y

the agar tube method.

The results are summarised in Table 4, grouping the samples ac-

cording to the indirect quantitative estimate. The two direct

methods agree well r = 0.77, P < 0.01; based on arc sin ~/per

cent). With 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 bu t one of less tha n 1 rhizobium per gram

were accompanied by reduced nodulation in both the field mean,

55 per cent) and core methods 65 per cent).

T A B L E 4

D i r e c t a n d i n d i r e c t d e m o n s t r a t i o n o f r h iz o b i a i n s ol l

% N o d u l a t e d P l a n t s

o il s g r a d e d b y p l a n t d i lu t i o n c o u n t

(per g so i l )

> 10

1 - 1 0

< 1

479

38

36

10

5

5

2

F i e l d [ C o r e

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 6

58 . 55 100 ~ 65

3 63

14 50

The degree of nodulatio n in the sown soll was more tha n one might

have expected from the counts. The roots of the plants from field

treatments with less than 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 then have

reflected an exploration of a greater depth of soff.

Re co ve ry at low er soi l d i lu t i on s . A large col lect ion of

samples from what appeared to be a sparsely popula ted soil was tested

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78 j .

A . T H O M P S O N A N D J . M . V I N C E N T

f o r t h e n o d u l a t i o n o f c l o v e r w i t h t h e a g a r t u b e m e t h o d , u s i n g fo u r in -

c r e a s in g d i lu t io ns e qu iv a l e n t t o 1, 0 . 1 , 0 . 01 , a n d 0 .001 g so i l pe r t ub e .

S i x t y - o n e s a m p l e s g a v e a t l e a s t o n e p o s i t i v e t u b e b u t s o m e o f t h e s e

t h o u g h p o s i t i v e a t h i g h e r d i l u t io n w e r e n e g a t i v e a t a l o w e r d i l u t i o n

m o r e s o i l) . I t w a s e s s e n t i a l t o a r r i v e a t a n a p p r a i s a l o f t h e s i g n if i-

c a n c e o f s u c h s k i p s .

T o f a c i l it a t e p r e s e n t a t i o n , t h e n e g a t i v e t u b e s h a v e b e e n c l as s if ie d

a s f o l lo w s a n d i l lu s t r a t e d w i t h tw o r e p r e s e n t a t i v e s a m p l e s T a b l e

5 A ) : Sk - n e g a t i v e t u b e s a t l e a s t o n e t e n f o l d d i l u t i o n b e l o w a

p o s i t i v e t u b e A 1 ) ; t w o t e n - f o l d d i l u t i o n s b e l o w A 2 ) ; Paired negative

- n e g a t i v e t u b e a t t h e s a m e d i l u t i o n a s t h e h i g h e s t p o s i t i v e t u b e

B ) ; Normal negative - n e g a t i v e t u b e a t o n e t e n - f o l d d i l u t i o n a b o v e

t h e h i g h e s t p o s i t i v e C ) o r a t t h e l o w e s t d i l u t i o n o f a w h o l l y n e g a t i v e

se r ie s D) .

O b s e r v a t i o n s o f n e g a t i v e t u b e s s h o w e d t h a t t h e s e e d l in g s c ar r i e d

r o o t h a i r s s o t h a t i n f e c t io n s i t e s w e r e a v a i l a b le . T u b e s w e r e t h e n

e x a m i n e d f o r t h e p r e s e n c e o f r h iz o b i a b y b r e a k i n g u p t h e a g a r a n d

t h e p l a n t , a n d t h o r o u g h l y s h a k i n g w i t h g la s s b e a d s a n d 1 0 m l s t e ri le

T A B L E 5

R e - t e s t o f c o n t e a t s o f 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 m p l e d e s i g n a t i o n o f n e g a t i v e t u b e s

W e i g h t o f s o il i n s u b - s a m p l e g )

S a m p l e R e p l i c a t io n

1 I 0 . 1 [ O .O 1 I 0 . 0 0 1

1 i ) + + - - B ) - - C )

i i) - - A ~ ) i - - A 1 ) + - - C )

2 i ) - -

D )

,] - - - - - -

i i ) - - D ) [ - - - - - -

B . R e - 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 N o d u l a t i n g [ N o 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 A 1

P a i r e d n e g a t i v e s B

N o r m a l n e g a t i v e s C

N o r m a l n e g a t i v e s D . . . .

2 2

9

1 2

9

5

4

1

6

1 5

3 O

4 6

1 1 4

9 6

6 0

4 5

2 3

1 0

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 o f a t le a s t 2 0 r h i z o b i a i n t h e s a m p l e d t u b e .

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water. Duplicate 0.5-ml aliquots were then 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 nor mal 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 di stribution

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 data 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 A B L E 6

R e e o v e r y o f r h i z o b i a f r o m p u r e c u l t u r e w i t h a n d w i t h o u t a d d i t i o n o f s o il t o a g a r t u b e s

A d d e d s o l l ( g / t u b e )

S t e r i l i se d ] U n s t e r i l e

0 0

0 0 . 0 1

0 1

1 0

1 0 0 1

1 1

T u b e s w i t h n o d u l e s fo r

S u c c e s s iv e F o u r f o l d D i l u t io n s *

L o g M P N

3 2 . 5 2

3 2 . 7 1

0 2 . 0 3

4 2 . 9 1

0 2 . 1 9

0 1 . 4 9

E x p e c t e d P o s i t iv 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 1 0 0 2 5

9 5 f i d u e ia l l i m i t s f o r l o g M P N o f e a e h t r e a t m e 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 . 6 1

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

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8 0 J . A . T H O M P S O N A N D J . M . V I N C E N T

r e l i a b i li t y e r e n w i t h t h e a d d i t i o n o f a f a i r l y l a r g e n u m b e r o f rh i z o b ia .

T w o f u r t h e r e x p e r i m e n t s g a r e s m a l l , t h o u g h n o t s i g n i f i c a n t , d e -

p r e s s io n s w h e n 1 g s ol l w a s a d d e d t o a g a r p l a n t - t u b e s u s e d i n t h e

r e c o v e r y o f c o u n t e d r h iz o b ia .

T a b l e 7 g i v e s r e s u l t s o f a m o r e c o m p r e h e n s i v e e x p e r i m e n t e x -

a m i n i n g r e c o v e r y o f A ) a c o u n t e d p u r e c u l t u r e a n d B ) a d i f f e r e n t

p o p u l a t i o n i n s oi l i ts e lf . T h e a d d i t i o n o f u n s t e r i l e s o ff c a u s e d a c o n -

s i s te n t d e p r e s s io n w h i c h w a s m o r e m a r k e d w h e n c o n t a m i n a t i n g s oil

m i c r o - o r g a n is m s h a d h a d a d d i t i o n a l t im e t o b e c o m e e s t a b li s h e d b e-

l o r e i n o c u l a t i o n A iii). T h e a d d i t i o n a l s o ff r e p r e s e n t e d in B i v) a p -

p e a r s t o h a v e e x e r c i s e d a f u r t h e r d e p r e s s i n g e f f e c t.

T A B L E 7

R e c o v e r y o f r h i z o b i a a f t e r t h e a d d i t i o n o f u n s t e r i l e s o ff t o a g a r t u b e s

S o ff a d d e d a t S o f f a d d e d 7 d a y s

R h i z o b i a l s o u r e e i n o c u l a t i o n b e f o r e i n o c u l a t i o n L o g M P N *

g) g)

A .

B .

i ) P u r e c u l t u r e

i i ) P u r e c u l t u r e

iii) P u r e c u l t u r e

i v ) P u r e c u l t u r e

i) S o il s u s p e n s i o n

ii) S o i l s u s p e n s i o n

iii) S o i l s u s p e n s i o n

i v) S o f f s u s p e n s i o n

v ) S o l l - i n - s o l l

v i ) So i l - i l l - so i l

q

0 . 5

0 . 5

0 . 5

0 . 5

0 . 5

0 . 5

0 . 5

0 . 5

0 . 5

0 . 5

0 . 5

1 7 7

1 3 7

0 . 8 8

0 . 8 8

> 4 . 2 6

3 . 9 2

3 . 6 8

2 . 9 9

2 . 1 3

1 7 7

* L e a s t s i g n i f i c a n t d i f f e r e n c e P = . 0 S) , 0 . 6 5 .

A n a t t e m p t t o s i m u l a t e t h e c o n d i ti o n o f h a v i n g f e w r h iz o b i a in

t h e s o ll, a n d h a v i n g t h e t o t a l s o ll r e c e i v e d b y e a c h t u b e c o n s t a n t ,

w a s m a d e d i l u t i n g t h e s o il b e i n g a s s a y e d i n r h i z o b i u m - f r e e so ff s u c-

c e s s iv e l y b y f ir s t b r e a k i n g t h e a g g r e g a t e s t h o r o u g h l y i n m o r t a r a n d

p e s tl e, a n d t h e n m i x i n g in t h o r o u g h l y w i t h o u t a d d i t i o n o f w a t e r

T a b le 7 , B v , v i) ). T h e r e c o v e r y w a s v e r y p o o r w h e n c o m p a r e d w i t h

t h e c o r r e s p o n i n g w a t e r - d i l u t e d se r ie s B iii a n d i v) . T h i s p o o r r e -

c o v e r y s e e m e d f ir s t t o i n d i c a te t h a t t h e r e h a d b e e n i n a d e q u a t e d is -

p e r s i o n o f t h e i n i t i a l s a m p l e i n s u c c e s s iv e d il u ti o n s . A n i n d e p e n d e n t

c h e c k d o n e w i t h t h e 1 0 a n d 1 0 0 s o i l - i n - s o i l d i l u t i o n s s h o w e d

r e a s o n a b le a g r e e m e n t b e t w e e n t h e m 90 0 a n d 150 r e c o v e r e d ). A p -

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D E T E C T I O N A N D E S T I M A T I O N O F R H I Z O B I A I N S O I 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)

A m o u n t o f s o il

l g

E x p e c t e d f r o m B i) T a b l e 7 ) . . 1 8 0 00

I n t e r n a l c h e c k 1 2 00 0

E x p e 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

1 8 0 0

1 2 0 0

8

8

0 l g ] . 0 0 l g

180 18

120 12

8 8

6 1

[ . 0 0 0 l g

1.8

1 .2

6

0

It seemed likely, therefore, that 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 al rea dy described. All were positive on re-test as was one of

two nor mal negat ives at dilution 5. Failure to suspend the rhi-

zobia in liquid suspension, had interfered with detection, probably

because of greater difficul ty in establishing contact with the root

surface.

D I S C U S S I O N

The present data 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 summarise d in terms of 11

comparisons with the plate counts and the lan t-dilution count th at

yielded the following average Iigures:

L o g p l a t e c o u n t I L o g p l a t e M P N L o g p l a n t M P N

4 . 1 8 ] 4 . 0 9 4 . 1 7

The presence of soil, however, can seriously affect the quanti tat ive

recovery of rhizobia. The difficu lty 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

th at a large proport ion 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, a nd

indicated rather tha t a sufIiciently unfavourable environment had

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8 2 J A T H O M P S O N A N D J M V I N C E N T

b e e n e s t a b l i s h e d i n t h e s k i p t u b e t o p r e v e n t n o d u l a t i o n . I t i s a l s o

q u i t e l i k e l y t h a t s o m e o f t h e s k i p t u b e s , i n w h i c h r h i z o b i a c o u l d n o t

b e d e m o n s t r a t e d b y f u r t h e r i s o la ti o n , h a d b e c o m e s u ff i ci e n tl y u n -

f a v o u r a b l e f o r t h e i r i n h i b i t i o n o r d e a t h .

P o s i t i v e e v i d e n c e o f t h e u n f a v o u r a b l e a c t i o n o f a r e l a t i v e l y l a r g e

a m o u n t o f u n s t e r i le s o il i s p r o v i d e d i n T a b l e s 6 a n d 7 . T h e i n t e rf e r in g

e f f e c t w a s a f u n c t i o n o f a m o u n t o f s o i l , i t s c o n t e n t o f o t h e r m i c r o -

o r g a n i s m s a r e l a t i v e l y s m a l l q n a n t i t y o f u n s t e r i l e so il .0 1 g ) b e i n g

a b l e t o c r e a t e a n u n s a t i s f a c t o r y t u b e e n v i r o n m e n t w i t h 1 g s t er i le

s o il ), a n d t h e l e n g t h o f t i m e t h e i n t e r fe r i n g p o p u l a t i o n h a d t o e s -

t a b l i sh i t s e l f Ta b le 7 ) .

T h e d i f f i c u l t y i n th e c a s e o f s o il s h a v i n g a t l e a s t a f e w h u n d r e d

r h i z o b i a p e r g i s n o t l i k e l y t o b e t o o s e ri o u s , i n t h a t h i g h e r d i l u t i o n s

a r e l i k e l y t o y i e l d p o s i t i v e s a n d j u d i c i o u s n o t i c e c a n b e t a k e n o f

s k i ps , e i t h e r q u a n t i t a t i v e l y o r i n p r e s e n t i n g a n d i n t e r p r e t i n g t h e

r e s u lt s . T h e r e a l p r o b l e m l ie s i n th e v a l i d i t y o f d e t e r m i n a t i o n s w i t h

v e r y l o w n u m b e r s f or w h i ch t h e d i lu t io n s e x p e c t e d t o b e p o s i ti v e

w o u l d h a v e t h e c o m b i n a t i o n o f a l a rg e a m o n n t o f s o il 1 -0 .1 g ) a n d

f e w r h i z o b i a .

T h e f i r s t a p p r o a c h t o t h i s i s t o s u b s t i t u t e f o r t h e a g a r t u b e a n

a s s e m b l y w h i c h m i g h t , b e c a u s e o f a m o r e p o r o u s s u p p o r t m a t e r ia l

a n d / o r i t s s iz e, p e r m i t l a rg e r s a m p l e s o f s o il t o b e a d d e d w i t h o u t

h a v i n g t h e s a m e u n d e s i r a b l e e f f e c t s . I n f a c t t h e t r e n d t o w a r d s s u -

p e r i o r i t y o f b o t t l e j a r s , a n d e v e n s a n d t u b e s , o v e r a g a r t u b e s , i n

T a b l e 3 w o n l d b e e n c o u r a g i n g w e r e i t n o t f o r t h e i r c o n s i d e r a b l e

i n f e r i o r i t y i n r e c o v e r i n g rh i z o b i a i n p u r e c u l t u r e t e s t s T a b l e 1).

B r o c k w e l l 1 a ls o f o u n d v e rm i c u l it e t u b e s u n d e r e s t im a t e d a p u re

c l o v e r p o p u l a t i o n .

T h e e x p e r i m e n t i n w h i c h l o w - c o u n t so il s w e r e s i m u l a t e d b y s o i l-

i n -s o i l d i l u t i o n T a b l e 7 B , v a n d v i ) n o t o n l y e m p h a s i s e d t h e d i f fi -

c u l t y o f r e c o v e r i n g a f e w r h iz o b i a i n a r e l a t i v e l y l a r g e a m o u n t o f s o ll

b u t a l s o a c c e n t u a t e d a p o s s i b l e s p a t i a l f a c t o r t h a t c o u l d d e l a y c o n -

t a c t b e t w e e n t h e f e w r h i z o b i a p r o b a b l y i n d i s c r e te m i c ro - c o lo n i e s)

a n d t h e r o o t s u rf a c e. T h e a d d i t i o n o f s u f f ic i e n t w a t e r w i t h t h e s o il

a n d s o m e d e g re e o f a g i t at i o n i n t h e t u b e w o u l d p r o b a b l y o v e r c o m e

t h i s p a r t o f t h e p r o b l e m .

F i n a l l y o u r e x p e r ie n c e e m p h a s i s e s t h e v a l u e o f d i r e ct , t h o u g h o n l y

s e m i - q u a n t i t a t i v e , m e t h o d s f o r t e s t i n g f o r r h i z o b i a in s p a r s e l y p o p u -

l a t e d s o ll s. S o il a m e l i o r a t io n , f r e e d o m f r o m c o n t a m i n a t i o n a n d

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mainten ance of adequate moisture will all be impor tant in such tests.

The fact that the rhizobium-free seedling will be able to explore a

reasonable volume of soll means that it can provide information on

the spatial distdbution 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 quantitative 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 importa nt difficult cases comp. Table 5B).

S U M M A R Y

T h 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 o r t h e e s t i m a t i o n o f r h i zo b i a , i n w h i c h

s m a l l - s e e d e d h o s t s a r e g r o w n o n a g a r w i t h i n t e s t - t u b e s , i s a p p l i c a b l e t o s o il s

w i t h a m o d e r a t e r h i z o b i a l p o p u l a t i o n i n t h e o r d e r o f a t l e a s t 1 0 0 /g ). A c c o u n t

m i g h t h a v e t o b e t a k e n o f

skips

l es s d i l u t e d : n e g a t i v e , w h e n m o r e d i l u t e d

a r e p o s i t i v e ) l i k e l y t o r e s u l t , a t l e a s t i n p a r t , I r o m u n f a v o u r a b l e c o n d i t i o n s f o r

r h i z o b i a l s u r v i v a l , m u l t i p l i c a t i o n o r n o d u l a t i o n . B e c a u s e o f s u c h e f fe c t s, a

s p a r s e p o p u l a t i o n i n t h e o r d e r o f 1 0/g ) m a y n o t b e d e t e c t e d e v e n w i t h o u t

d i l u t i o n 1 g s o il p e r p l a n t t u b e ) . L o c a l i s a t i o n o f r h i z o b i a i n t h e s o i l i s l i k e l y

t o b e i m p o r t a n t i n d e t e rm i n i n g c o n t a c t w i t h t h e p l a n t r o o t s in t h e d i lu t io n

c o u n t a n d i n s a m p l i n g f r o m t h e f ie ld . D i f fi c u l t i es w i t h s p a r s e ly p o p u l a t e d

s o il s c a n b e p a r t l y o v e r c o m e b y c a r e f u l l y c o n d u c t e d d i r e c t s o w i n g s o f s t e ri l is e d

s e e d , p r e f e r a b l y i n t h e C O llI in es o f co r es , e i t h e r l e i t i n t h e f i e l d o r b r o u g h t b a c k

t o t h e g l a s s b o u s e .

A C K N O W L E D G M E N T

T h e w o r k r e p o r t e d i n t h i s p a p e r h a s b e e n s u p p o r t e d b y r e s e a r c h fu n d s

f r o m t h e U n i v e r s i t y o f S y d n e y , t h e R u r a l C r e d i ts D e v e l o p m e n t F u n d o f t h e

R e s e r v e B a n k o f A u s t r a l i a a n d b y W h e a t I n d u s t r y R e s e a r e h F u n d s .

Received October 25, 1965

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8 DETECTION AND ESTIMATION OF RHIZOBIA IN SOIL

REFERENCES

1 Br oe kw el l, J., Accuracy of a plant-infection technique for counting populations of

R h i z o b i u m t r i] o l ii . Applied 3/licrobiol. 11, 377-383 (1963).

2 Da te , R. A. and Vin ee nt , J. M., Determination of the numb er of root-nodule

bacteria in the presenee of other organisms. Australian J. Exp. Agr. Animal Husband-

ry 2, 5-7 (1962).

3 Fi s h er , R. A. and Ya te s, F., Statistieal Tables for Biologieal, Agrieultural and

Medical Researeh. Oliver and Boyd, London (1953).

4 Gi bb on s, F. R. and Ha ll sw or th , E. G., Map of the soils of the Namoi Region.

I n The Namoi Region. A prelimi nary Sur vey of Resonrees . New South Wales

Premier s Dept. (1950).

5 Ha lv or se n, H. O. and Ziegl er, N. R., Applieation of statisties to problems in

bacteriology. I. A means of determining baeterial populations by the dilution

method . J. Baeteriol. 25, 101-121 (1933).

6 Hel y, F. W., Be rg er se n, F. J. and Br oe kw el l, J. , Mierobial antagonism in the

rhizosphere as a faetor in the failure of inoeulation oI subte rran ean elover. Australian

J. Agr. Research 8, 2 4 4 4 (1957).

7 Je ns e n, H. L., Nitrogen fixation in leguminous plants. I. General eharaeters of root

nodule baeteria isolated from speeies of M e d i c a g o and T r i I o l i u m in Australia. Proc.

Linn. Soc. N.S.W. 66, 98-108 (1942).

8 Le on ard , L. T., A simple assembl y for use in testing cultures of rhizobia. J. Baeteriol

4~

5 2 3 5 2 5 (1943).

9 M a r q u e z , R. G., Studies of the Root Nodule Bacteria. Thesis, M.Se. Agr., Univ.

Sydney (1956).

10 Pu re ha se , Hi la ry F. and Nu tm an , P. S., Studies of the physiology of nodnle

formation. VI. The influenee of bacterial numbers in the rhizosphere on nodnle

initiation. Ann. Botany 21,439-454 (1957).

11 Tu zi mu ra , K. and Wa ta na be , I. , Estimation of number of root-nodule baeteria

by a nodulation-dilution frequeney method. Eeologieal stndies of

R h i z o b i u m

in solls.

(Part I). Soff Sci. Plant Nutr. 7, 61-65 (1961).

12 Vi nc en t, J. M. and Cro ft s, F. C., Pasture improveme nt in the Pilliga. Rep. No. 3,

School of Agriculture, Univ. Sydney (1958).

13 Wi ls on , J . K., Legume baeteria popula tion of the soil. J. Am. Soc. Agron. 18,

9 1 1 - 9 1 9 1 9 2 6 ) .

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