BIOLOGIGAL C O N T R O L OF W H I T E R O T OF O N I O N

II. EFFECTIVENESS OF PENIGILLIUM NIGRICANS (BAIN.) THOM

by

A B D U L GHAFFAR 1)

Department o/Botany, The University, Karachi, Pakistan

(with 9 figs.)

(14.X.1968)

INTRODUCTION

In a previous paper (4) Penicillium nigricans (BAIN.) THOM WaS

found to inhibit the growth of Sclerotium cepivorum BERK., on agar
plate, inhibition taking place due to diffusion of toxic metabolite
into the medium. When soil cultures of P. nigricans were added to
garden loam, a pronounced control of white rot infection was ob-
tained. The present paper describes the persistence of this effect of
P. nigricans on the control of white rot disease.
The isolates of S. cepivorum and P. nigricans were the same as
used before (4). The bunching salad onion, var. 'white lisbon', in
its second true leaf stage was used throughout the experiments.

EXPERIMENTAL

Effectiveness of P. nigricans cultures added to garden l o a m

in controlling infection of onion seedlings by S. c e p i v o r u m
Ex2bt. 1
Garden loam, p H 7.2, amended with 5 % maize meal, adjusted to
50 % w.h.c. (7), was sterilized in 100 g quantities in 250 ml Erlen-
meyer flasks for one hour at 20 lbs/in ~. Each flask after inoculation
with 3 ml of a conidial suspension (app. 1 × 106 conidia/ml) collected
from 10 day old colonies of P. nigricans on Czapek-Dox yeast agar,
was incubated for 3 weeks at 22 ° C. These cultures were thoroughly
mixed with unsterilized sieved garden loam to give 1 : 4 parts of

1) This work was carried out at tile D e p a r t m e n t of Botany, The University,

Birmingham, England. I wish to ttiank Prof. C. J. HICKI~iAN for his invaluable
advice and encouragement a n d to Dr. P. W. :BRIAN for the gift of griseofulvin.
114 A, G~IAFFAR

inoculum : soil (w/w). Controls were kept in which, (i) sterile soil
maize meal, on which no fungus had grown, was similarly diluted,
and (ii) in which unamended garden loam was used. The soils were
transferred to 3.5" pots, 250 g per pot. Onion seedlings inoculated
at the bulb base with 5 mm diameter disks of S. c@ivorum inoculum
were transplanted in each pot at intervals of 0, 1, 5, 10, 20 and 30

lO0~ '-A A &

9O

8O

70

o o

5C A

3C 0

2O

lo
\
O

0 5 10 20 30
DAYS

A GA/~DEN L O A M + p.ni~ricams
KEy 0 + SOIL/~,IAIZE M E A L

• , C OhmfRe L

Fig. I. Control of white rot of onions in garden loam alone and those mixed with
Penicillium nigricans or soil maize meal. (Expt. 1).

days, keeping 2 pots for each treatment with 5 seedlings per pot.
The pots were kept on a green house bench ( ~ 20 ° C) and main-
tained at 40 % w.h.c, by watering daily. Observations on the infec-
tion of white rot were recorded at the end of 2 weeks from each
transplanting (Fig. 1). In the series where P. nigricans was added
no seedlings wilted in soils which had an incubation of up to 5 days
 BIOLOGICAL CONTROL OF WItlTE ROT OF ONION I I ] 15

A. WIIITE L'OT
IO0~L

90 @
80 &

70

o 6o

x~.4o

3o /it

2O

5 lo 20 3o 40
DAYS
B, G]~ISEO~ULVIN
100 I

9o
A GARDEN LOAM + P.nigricans (IJnau~oelaved)
80,~ ,
l~y • " * " (Autoela'ved)
% O " . SOZL/MaZZE ~ A L
7c
6C ~ • " , CONTROL

o 5c

l,o!
~3o
2o
10

5 to 20 30 4Q
DAYS

Fig. 2. Control of white r o t of onions and c o n c e n t r a t i o n of griseofulvin in garden

loam alone and those mixed w i t h autoclaved or u n a u t o c l a v e d cultures of P. nigricans
or soil maize meal. (Expt. 2),

before transplanting. Where the incubation period before trans-

planting was 10 days, 50 % of the seedlings were infected whereas
in treatments with 30 days incubation no infection occurred. Garden
loam alone did not provide any control. Garden loam supplemented
with soil maize meal produced a progressive control of white rot,
80 % of the seedlings remaining healthy where incubation period
before transplanting was 10--20 days, whereas, in treatments with
30 days incubation only 10 % of the seedlings remained healthy.
Expt. 2
A similar experiment was made to examine whether the control
associated with P. nigricans was due to the presence of griseofulvin,
116 A. G~AFFAR

an antibiotic which it is known to produce (2). Cultures of P. nigri-

cans were added to garden loam as before. In another treatment P.
nigricans cultures on soil maize meal, autoclaved at 15 lbs/in ~ for
15 minutes, being similarly diluted with garden loam was included
since griseofulvin can be autoclaved without loss of effect (2). The
soils were transferred to pots, kept in a greenhouse (:L 20 ° C) and
maintained at 10 °/o w.h.c, by watering every day. Onion seedlings
inoculated with S. cepivorum were transplanted to different soils
at intervals of 0, 5, 10, 20, 30 and 40 days keeping 2 pots for each
treatment and 5 seedlings per pot. Observations on the infection of
white rot were recorded after 2 weeks of transplanting (Fig. 2A).
There was no difference in the results where autoclaved or un-auto-
claved cultures of P. nigricans were used. The inoculum having been
in the soil for a period of 5 days produced 100 °/o control of white rot.
Where the incubation period before transplanting was 10 days, this
effect was reduced. After an incubation period of 20 days a second
increase in control was recorded. Garden loam alone produced no
control throughout the experiment. Where garden loam was sup-
plemented with sterile soil maize meal, a progressive control of the
disease was obtained. This, however, was not a permanent effect as
the control declined after reaching a maximum after 20 days de-
composition of the maize meal.
At the same intervals of 0, 5, 10, 20, 30 and 40 days, the amount
of griseofulvin present in each soil was estimated (11). Twenty g
(dry wt.) soil samples were extracted with ether in a Soxlet appara-
tus for one hour. The ethereal extract was evaporated to dryness
and the residue dissolved in 2 ml ethanol which was made up to
20 ml in McIlvaine's buffer adjusted to p H 3.5. This extract was
assayed for antifungal activity b y a bio-assay technique using
Botrytis allii as test organism (3). The amount of griseofulvin present
in soil was estimated b y comparing the degree of coiling and distor-
tion of tile hyphae of B. allii produced by soil extracts with the
effects produced b y known concentrations of griseofulvin. (Fig. 2B).
Where P. nigricans was used the amount of griseofulvin was 80 #g/g
of soil during a period of first 5 days of incubation. Soil samples
taken at subsequent intervals of the experiment showed a decline
in the concentration of griseofulvin. Garden l o a m alone or that
supplemented with soil maize meal had no effect.
B y comparing Fig. 2A with Fig. 2B it would appear that the
initial control of white rot was somewhat related to the amount of
griseofulvin present in the soil. With a decline in concentration of
griseofulvin there was a decline in the control of the disease. Later
a secondary control of the disease was observed without any increase
in the concentration of griseofulvin. As this effect was more pro-
nounced in soil amended with soil maize meal it suggested that it
might be of microbial origin. The experiment was therefore repeated
to elucidate this point.
 BIOLOGICAL CONTROL OF WHITE ROT OF ONION II 117

Expt. 3
The organization of the experiment was the same as before.
Cultures of P. nigricans or sterile soil maize meal added to garden
loam in 1 : 4 ratio were stored in 2 lb biscuit tins covered with lid
and maintained at 40 % w.h.c. Ordinary garden loam, similarly

A. W H I T E ROT
loo, --A .......... A - - A

90

8O

g 70

5o

4o

3o

2 5 7 10 1/4 18 21 25 23 33
DAYS

B. G R I S E O F U L ¥ I N
lOO

90

80

7o
--~ll
•'"~'----~ GARDEN LOAM + P . n l g r i c a n s
,q KEy O " + SOIL/MAIZE 31EAL
6c
O " , CONTROL

O~ 5C

30

2o A

,,A
o 2 5 7 lO 14 18 21 25 ;~9
DAYS

Fig. 3. Control of white rot of onions and concentrations of griseofulvin in garden

loam alone and those mixed with cultures of P. nigricans or soil maize meal. (Expt. 3).

stored, was used as control. At intervals of 0, 2, 5, 7, 10, 14, 18, 21,

25, 29 and 33 days, samples of soils from the different series were
taken and observations on the following were obtained:
1. Effect on white rot; b y transplanting seedlings inoculated with
S. cepivorum into pots of soil at 40 % w.h.c., (Fig. 3A).
118 A. GHAFFAR

2. Concentration of griseofutvin; by extracting soil samples with

ether in a Soxlet apparatus, followed by bio-assay with B. allii
spores (Fig. 3B).
3. Population of fungi, bacteria and actinomycetes was obtained
by dilution plate technique on Czapek-Dox yeast agar (4).
The average number of colonies from 3 plates was multiplied
by the dilution factor to obtain the number of microorganism
per g of the soil sample (Fig. 4).
4. Number of bacteria and actinomycetes antagonistic to S. cepi-
vorum on agar plate. For this random samples of bacteria and
actinomycete colonies appearing on dilution plates were in-
oculated around an actively growing colony of S. cepivorum on
Czapek-Dox yeast agar at pH 6.9. There were 25 samples from
each treatment, 5 isolates per plate (Fig. 4).

~i FUNGI

0
~ BACTE}{IA
2 5 7 10 14
DAYS
18 21 25 29
l
33

~o 01,.~
6 ~ .5 7 lo 14 ~8 2~ 25 ~9 33
DAYS

I0 ACTIN0)fYCETES

. .

0
. .

2 5 7
J
10 14
_
18 21
J
25 29
d
33
DAYS

D GARD~3N LOA}[
KEY ~ " + P.ni~ricans
" + S O I L / ~ I Z E MEAL
I BACTER!A ANTAGONISTIC TO S,oepivorum

Fig. 4. Population of fungi, bacteria and actinomycctes in garden loam alone and
those mixed with cultures of P. nigricans or soil maize meal. (Expt. 3).
 B I O L O G I C A L C O N T R O L OF W H I T E ROT OF ONION II 119

The control of white rot and its relation with the concentration of
griseofulvin was similar to what was obtained before. In the treat-
ment where P. nigricans was used, the number of Iungal colonies
appearing on the dilution plates varied between 20--24 × 107 per g
of dry soil (Fig. 4). These were all found to be P. nigricans. The
number of bacterial colonies when sampled at successive intervals
however, showed a gradual increase in number with a fall towards
the end of the experimental period. It is interesting to note that the
number of bacteria (species of Bacillus and Bacterium) antagonistic
to S. cepivorum do also shout a rise and fall similar to the results
obtained on the control of white rot disease, although they do not
correspond at the same time intervals. Another interesting observa-
tion made in this experiment was the appearance of Pseudomonas
sp. on the dilution plates prepared from the soil where P. nigricans
was used. Such a Pseudomonas sp. has previously been reported in
the degradation of two griseofulvin derivatives (12).
Expt. 4
From the previous experiment it seemed that the population of
P. nigricans in soil remained more or less uniform as depicted with
the results of dilution plates (Fig. 4). The concentration of griseo-
fulvin, however, declined. In order to see whether with further addi-
tion of maize meal, P. nigricans can be stimulated to grow so as to
prolong its effectiveness, another experiment was carried out which
included the following treatments:
1. Twenty days old cultures of P. nigricans mixed with unsterile
garden loam in the proportion of 1 : t;
2. as no. 1; supplemented with 4 % maize meal;
3. sterile soil maize meal mixed with unsterile garden loam, 1 : 4;
4. as no. 3; supplemented with 4 % maize meal;
5. ordinary garden loam was used as control.
The above soils adjusted and maintained at 40 % w.h.c, were
stored in 2 lb biscuit tins and kept in the green house ( ± 20 ° C).
Soil samples at intervals of 0, 2, 5, 7, 10, 14, 18, 21, 24 and 28 days
were taken and observations on the controi of white rot, concentra-
tion of griseofulvin and the population of microorganisms were ob-
tained as before. The results from treatments 1, 3 and 5 showed a
similar pattern to t h a t obtained in the previous experiments. Addi-
tion of 4 ~/o maize meal to garden loam inoculated with P. nigricans
prolonged the effectiveness of the fungus on the control of white rot
when compared with soil to which no maize meal was added (Fig. 5).
The number of P. nigricans colonies appearing on dilution plates
made from the inoculated garden loam to which 4 % maize meal
was added, showed a gradual decline when compared with its con-
trol, (Fig. 6). During this prolonged effective period the concentra-
tion of griseofulvin however remained at 80 #g/g of dry soil. This
concentration of griseofulvin later declined and with its decrease a
120 A. GHAFFAR

decline in white rot control followed. Although no subsequent in-

crease in griseofulvin concentration took place, a second period of
control of white rot disease was obtained which was related to the
increase in number of bacteria antagonistic to S. cepivorum (com-
pare Figs. 5 and 6).

A, W H I T ~ R O T

8C

7c
!to
~,. 5C

w w
10 21 2lI~
DAYS
B. G R I S E O F U L V I N
100 r A GARDEN LOAM + P.ni~ricans
! KEY • . + ,,

90[ + 4% ~t~izE ~ A L
|. . . . . A A • ,, , CONTROL
8 0 Ame-.~ic---~gw---

o 2 .s 7 ,o 14 18 21 24 28
DAYS

Fig. 5. Control of white rot of onions and concentration of griseofulvin in garden

loam alone and those mixed with cultures of P. nigz{cans or P. n{gricans plus 4 ~to
m a i z e meal. (Expt. 4).

Another interesting result of this experiment (treatments 3, 4 and

5) was that mere addition of maize meal to garden loam produced
a progressive control of white rot compared with garden loam alone
which had no effect (Fig. 7). Where a total of 5 % maize meal was
 BIOLOGICAL C O N T R O L OF WHITE ROT O F ONION II 121

FUNGI

'i I
oi
5~
0
BACTERIA
~O 14 DAYS 18 ;21 24
t
28

3¸
ox

__JJo ~ 5 10 i4 DAYS
18 21 24
J
28

. ACTINOMYCETES

o 2 5 7 ~o ,~ ,s xl 2~
_-]GARDEN LOAM
KEY ~ " + "P,ni~ricans
U " ÷ + 4% ~*AiZE ~ A L
U BACTERIA ANTAGONISTIC TO S. oepivorumm

Fig. 6. P o p u l a t i o n of fungi, b a c t e r i a a n d a c t i n o m y c e t e s in g a r d e n l o a m a l o n e a n d
t h o s e m i x e d w i t h c u l t u r e s of P . nigricans or /J. nigricans p l u s 4 ~o m a i z e meM.
( E x p t . 4).

used 90 % control of the disease was obtained in soil samples taken

after 14 days decomposition of the maize meal. There was a pro-
gressive increase in the microbial population (Fig. 8). The fungi
(mostly Geotrichum candidum and Mucorales) were non-antagonistic
to S. cepivorum on agar plates. The number of antagonistic bacteria
(species of Bacillus and Bacterium), however, showed an increase in
activity during the period of control, maximum population being
followed by maximum control of the disease (compare Figs. 7 and 8).
122 A. O~AF~AR

Effect of g r i s e o f u l v i n on S. c e p i v o r u m
(i) In agar culture
The requisite amount of griseofulvin was dissolved in 2 ml of
ethanol and made up to 100 ml in Mcllvaine's buffer at pH 4.4.
This was further diluted in buffer solution to get a series of con-
centration of griseofulvin, 20 ml of which was mixed to 20 ml of 2N
sterilized Czapek-Dox yeast agar at the same pH. The medium was
poured into 90 mm Petri dishes, in approximately 15 ml quantities,
there being 2 replicates at each concentration. Controls were kept
Ioo

9o

8O

7o

~ 60
"~ 5o
4o
3O

2o

lo

o 5 7 ao 14 18 21 24 28
DAYS

X GARDEN LOAM + M A I Z E MI~AL 5%

@ , CONTROL

Fig. 7. Control of w h i t e r o t of o n i o n s in g a r d e n l o a m alone a n d g a r d e n l o a m a m e n d e d

w i t h 1 or 5 % m a i z e meal. (Exl0t. 4).

which did not include antibiotics. The dishes were inoculated in the
centre with 5 mm disks of S. cepivorum inoculum and incubated at
22 ° C. Rate of growth of the fungus was measured daily up to a
period of 10 days (Fig. 9). S. cepivorum did not grow in media con-
taining 400 #g/ml of griseofulvin up to a period of 5 days after which
it grew slowly to produce a colony of 10 mm in 10 days time. Lower
concentrations of griseofuhdn retarded the growth of the fungus.
This fungistatic effect of griseofulvin on S. cepivorum is similar to
t h a t observed by BRIAN (3), AYTOUN (1), NAPIER et al. (10) with
other fungi.
(ii) In soil
Griseofulvin in 160 rag q u a n t i t y was dissolved in 2 ml of ethanol
and made up to 400 ml with tap water. This was further diluted to
get concentrations of 400, 200, 100, 50 and 10/~g of the antibiotics
 BIOLOGICAL C O N T R O L OF V I H I T E ROT OF O N I O N I I 123

FUNGI
3

.5 7 10 14 18 21 24 28
O
5 BACTERIA

o~
o
o 2 5 10 14 18 21 24 28

I01 ACTINOMYCETES

olr~h
l _

O 2 5 7 10 ~ t8 21 24 28

[--~ GARDEN LOAM

~Y ~] " + 17~ iqAIZE ~[EAL

B BACTERIA ANTAGONISTIC TO S. cepivorum

Fig. 8. Population of fungi, bacteria and &ctinomycetes in garden loam alone and
garden loam amended with I or 5 % maize meal. (Expt 4).

per ml of water. Fifty ml of these solutions were watered on to 250 g

of sieved garden loam (pH 7.1) in 3.5" pots so as to adjust it at 40 %
w.h.c. (7) thereby obtaining 80, 40, 20 and 2 #g of griseofulvin per g
of dry soil. There were 4 pots at each of the concentrations of the
antibiotics. Controls were kept in which no griseofulvin was used
in the soil.
Onion seedlings inoculated at the base of the developing bulb with
5 mm disks of S. c@ivorum inoculum from an actively growing
colony on Czapek-Dox yeast agar were transplanted to treated pots.
A second batch of seedlings, uninoculated with S. ce~ivorum were
 124 A. G~AFFAR

similarly transplanted to see the phytocidal effects of griseofulvin,

if any. There were 10 seedlings per treatment, 5 per pot. The pots
were kept on a green house bench (:k 20 ° C) and maintained at
40 % w.h.c, with tap water. After 2 weeks 60 % of the seedlings
developed white rot infection in soil containing 80 s g of griseofulvin
• CONTROL
90

8o

. / . 0.2 ~/~,z
7o

6o • J" .0o5~ / m l

5O /7
//
.
./j
/ j J /
j,.o,.,.
4o
//
30
0

20
///>i- 1-Jj.--f i - .2-,-"
10

I I I I I I I I I I

1 2 3 ~ 5 6 7 8 9 ~o

DAYS
Fig. 9. R a t e of g r o w t h o5 5clerotium cepivorum o n C z a p e k - D o x y e a s t a g g r c o n t a i n i n g
d i f f e r e n t c o n c e n t r a t i o n of griseofnlv~n (/zg/ml) a t 22 ° C.

TABLE I
Development o/white rot in/ection o/onion seedlings in garden loam containing diMerent
concentrations o/griseo/ulvin.

Concentration No. of seedlings wilted* F i n a l infection

of griseofulvin O/o
(#g/g of soil) T i m e in d a y s 7 10 16

0 10 10 10 100
2 10 I0 10 100
10 8 10 10 100
20 2 10 10 100
40 1 10 10 100
80 0 6 6 60

*) O b s e r v a t i o n s b a s e d on 6 s e e d l i n g s p e r pot.
 BIOLOGICAL CONTROL OF W H I T E ROT OF ONION t i 125

per g of soil. In series containing lower concentrations GI griseofulvin

although all the seedlings wilted, but the appearance of infection
was delayed (Table I). There was no observable deleterious effects
on the seedlings grown in soil containing griseofulvin.

CONCLUSION
In the experiments described in this paper, P. nigricans gave
consistently good results in the control of white rot disease of onion.
Twenty days old cultures of P. nigricans on soil maize meal, diluted
up to 0.25 strength with unsterile garden loam remained effective
up to at least 5 days. Further addition of maize meal prolonged its
effectiveness due to the growth of the fungus using organic sub-
strates. The control of white rot was related to the concentration of
griseofulvin in the soil, control declining with the decrease in its
antibiotic concentration. This decrease in griseofulvin concentration
can be explained by the antagonism of soil microorganisms towards
P. nigricans (11) and also because of rapid disappearance of griseo-
fulvin due to biological degradation (5, 12). Analysis of microbial
population of the soils inoculated with P. nigricans showed the
appearance of Pseudomonas sp. on the dilution plates. It m a y be
mentioned that such a Pseudomonas sp. has been reported to be
responsible in the inactivation of 2 griseofulvin derivatives (12).
An interesting observation made during the experiments was that
after the first decline in the control of white rot corresponding with
the decrease in the concentration of griseofulvin, a second period of
control of the disease was obtained without any corresponding in-
crease in the concentration of griseofulvin in the soil. This suggested
an effect due to some other microorganisms (species of Pseudomonas,
Bacillus and Bacterium) which increased in population during the
second period of control. A few of these inhibited the growth of S.
cepivorum in plate culture and also in soil, the Pseudomonas sp.
having no effect. It would appear that although Pseudomonas is
believed to be concerned in the degradation of griseofulvin (12), the
second period of control of the disease was brought about by the
increase in population of other antagonistic bacteria.
Griseofulvin, in pure form, showed fungistatic effects on the
growth of S. cepivorum in agar culture. At 400/~g of griseofulvin per
ml of the medium, S. cepivorum grew slowly after an interval of
several days. A similar effect was observed in soil, the appearance
of white rot infection being delayed in the presence of griseofulvin.
In the present study, where P. nigricans cultures were used (esti-
mated concentration of griseofulvin being 80 #g/g of soil) no infec-
tion occurred, although the concentration of gris,eofulvin later de-
clined. It would appear that this control could not be due to the
presence of griseofulvin in soil because of two opposing processes
occurring simultaneously: (i) the disappearance of griseofulvin in
soil (5, 12) when, (if) S. cepivorum should be continuing to grow
126 A. GHAFFAR

slowly in the presence of diminishing concentrations of this anti-

biotic. It would therefore suggest that a fungistatic effect on S. cepi-
vomm is effected b y griseofulvin, a complete control of the disease is
accomplished due to the effects brought about b y the increase in
population of antagonistic bacteria in soil.
These experiments also provided evidence that mere additions of
maize meal to soil had a pronounced effect on white rot control.
This was again accompanied by an increase in population of anta-
gonistic bacteria. Similar observations have been made on the
control of Phymatotrichum omnivorum (8, 9) and Sclerotium rol/sii
(6). It would therefore be interesting to study the effects of organic
amendments as possible means of biological control of white rot
disease.

Summary

A study has been made of the persistence of the effects of Penicil-

lium nigricans, in garden loam, on the control of white rot disease
of onion. Twenty days old cultures of P. nigricans, diluted up to
0.25 strength, remained effective up to 5 days. Further additions of
maize meal to the soil prolonged its effectiveness. The control of
white rot was associated with a concentration of 80 ~g of griseo-
fulvin per g of soil. Loss o5 activity in controlling white rot was
related to decline in griseofulvin concentration, which in turn was
associated with the build of Pseudomonas sp. in soil. Pseudomonas
had no effect on S. cepivorum. Decline in control b y P. nigricans was
followed by a second period of control which was related to increase
in number of bacteria antogonistic to S. cepivorum.
In agar culture griseofulvin produced a fungistatic effect on the
growth of S. cepivorum. Similarly in the presence of griseofulvin in
soil, the appearance of white rot infection was delayed. Evidence
was obtained to the fact that a fungistatic effect on S. cepivorum
is effected b y griseofulvin, complete control of white rot was related
to increase in population of microorganisms antagonistic to S. cepi-
vofum.

References
1. AYTOUN, R, S. C. 1956. T h e effects of griseofulvin o n c e r t a i n p h y t o p a t h o g e n i c
fungi. A n n , Bot., N.S. Lond., 2 0 : 297---305.
2. BRIAN, P. W~. 1949. S t u d i e s on t h e biological a c t i v i t y of griseofulvin. A n n . Bot.,
Lond., 13: 5 9 - - 7 7 .
3. BRIAN, P. W. • HEMMING, H. G. 1945. Gliotoxin, a f u n g i s t a t i c m e t a b o l i c
p r o d u c t of Trichoderma viride. A n n . appl. Biol. 3 2 : 214--220.
4. GHAFFAR, A. 1969. Biological c o n t r o l of w h i t e r o t of onion. I. I n t e r a c t i o n s of soil
m i c r o o r g a n i s m s w i t h Sderotium cepivorum BERg. M y c o p a t h . et Mycol. appl.
38: 3 8 - - 4 8 .
5. JE~ERYS, E. G. 1952. T h e s t a b i l i t y of a n t i b i o t i c s in soils. J. G e m MicrobioI.,
7: 2 9 5 - - 3 1 2 .
6. JOHNSON, S. P. 1953. S o m e factors in t h e c o n t r o l of t h e s o u t h e r ~ b l i g h t o r g a n i s m
Sclerotium rolfsii. P h y t o p a t h o l o g y , 4 3 : 363--368.
 BIOLOGICAL CONTROL OF WHITE ROT OF ONION I I 127

7. KEEN, B. A. 6; RACZKO%VSHI, H. 1921. The relatiol~ between the clay content

and certain physical properties of a soil .j. agric. Sci., I I: 441--449.
8. KING, C. J., HOPE, C. & EATON, E. D. 1934. Some microbiological activities
affected in manurial control of cotton root rot. J. agric. Res. 49: 1093---i107.
9. MITCHELL, R. B., HOOTON, D. R. 6; CLARK, F. E. 1941. Soil bacteriological
studies in the control of Phymatotrichum root rot of cotion. J. agric. ~Res., 63:
535--547.
1O. NAPIER, ]~UNICE j., TURNER, DOROTHY I. & RHODES, A. 1956. The in-vitro
action of griseofulvin against pathogenic fungi of plants. Ann. Bot. N.S., Lond.,
20: 461--466.
ii. WRIGHT, ]. M. 1955. The production of antibiotics in soil. II. Production of
griseolulvin by Penicillium nigricans. A n n . a p p l . B i o l . 4 3 : 2 8 8 - - 2 9 6 .
12. WRIGHT, J. M. • GROVE, J. F. 1957. The production of antibiotics in soil. V.
Breakdown ol griseofulvin in soil. Ann. appl. Biol. 45 : 36--43.