Egypt. J. Agric. Res. , 70 (2) , 7 99 7
PSEUDOMONAS L/NDBERG/1 AND CONIOTHYRIUM MINITANS AS BIOCONTROL AGENTS
EFFECTIVE AGAINST SOME SOIL FUNGI PATHOGENIC TO PEANUT
K. K. SABET1
M. A. MOSTAFA 1 OM-HASHEM I. EL-BANA 1
EBTISAM M. EL-SHERIF2
7 Faculty of Agriculture, Cairo University, Giza, Egypt.
403
2 Plant Pathology Research Institute, Agricultural Research Centre, Giza, Egypt.
(Manuscript received 6 May 1 990)
Abstract
Pseudomonas lindbergii Lindberg and Coniothyrium minitans
Campabell, were tested as biocontrol seed - treatment against peanut
damping-off and pod-rot organisms. P. lindbergii was more effective in
preventing pre- and post emergence damping-off. C. minitans, though
not particularly effective against seedling infection, showed noticeable
activity against pod-rot causal organisms. The fungicides showed some
effectiveness in protecting the seedling or the pods against infection but
not as much as the bacterial treatment.
INTRODUCTION
Several fungi were reported to infect the roots and pods of peanut. Zaher
et al. ( 1 9 84) found that Rhizoctonia so/ani, Fusarium oxysporum and Sclerotium
rolfsii were the most dominant fungi. They were highly pathogenic to peanut
causing pre- and post - emergence damping-off. Seed treatment with a biocontrol
404 K. K. SABET et al.
agent has been tried by some authors using Trichoderma harzianum.
The purpose of this work is to compare the effect of seed treatment with
biocontrol agents Pseudomonas lindbergii Lindberg and Coniothyrium minitans
Campbell with that of chemical treatment on pre- and post - emergence damping-off,
pod-rot and the yield of peanut.
P. lindbergii has been chosen since it produces an antibiotic lethal to a wide
range of fungi including Fusarium and Rhizoctonia (Lindberg, 1981 ). This antibiotic
was identified as the seven-membered ring compound tropolone (2-hydroxy-2, 4,
6-cycloheptatriene-1 -one) (Lindberg et al. 1980). lt was used as biocontrol agent
against Fusarium graminearum by Sabet ( 1987).
C. minitans was discovered by Campbell ( 1 94 7) in California on sclerotia
formed in cultures of Sc/erotinia sc/erotiorum. it was later isolated by Tribe
( 1 9 57) from sclerotia of Sc/erotinia trifoliorum and tested as a sclericide to
control onion white - rot caused by Sclerotium cepivorum (Ahmed and Tribe
1977). lt was not pathogenic to any of the test plants and could be used as a
biocontrol agent (Turner and Tribe 1976).
MATERIALS AND METHODS
In -vitro studies were done to investigate the antagonistic effect of P.
lindbergii and C. minitans, against F. oxysorum, R. so/ani and S. rolfsii. The
bacterium was streaked on PDA plates opposite to a 5 mm disk of the pathogen's
mycelial growth at 5 cm distance and incubated at 27 °C for one week. Effect of C.
minitans was evaluated in two ways : 1) The viability of fungal mycelium grown in
the contant region of both the fungus and C. minitans as well as non-contant region
(near the inoculum disk of the tested fungus), were determined by transfering disks
from these areas to new PDA plates. 2) Pycnidia of C. minitans were collected
from PDA plates and used to surface infest sclerotia of S. ro/fsii by dusting. Treated
propagules were placed on water agar plates, 1 0 sclerotia per dish. Control was
uninfested propagules. Th~ sclerotia used in this experiment were either formed on
PDA plates or on sorghum substrate. After 4 weeks of incubation at 20 °C, they
were collected, surface sterilized with 3 % sodium hypochlorite for 1 min, plated on
BIOCONTROL - PEANUT - SOIL FUNGI 405
PDA plates containing 30 ppm rose bengal, before autoclaving and 1 0 ppm CID's
Tetracid (oxytetracyclin hydrochloride, water soluble), after autoclaving, and
incubated at 25 °C. Viability and germination were observed after 1 week. This
method was a modification for that used by Trutmann et al. (1980) .
Field study was based on two years experiment (1987 and 1988) at two
locations (in Giza, Faculty of Agriculture Cairo University and Agricultural Research
Centre). To prepare the inoculum of P. lindbergii, it was grown on potato dextrose
broth PDB. Fifty ml of liquid media were added to each 1 OOml Erlenmeyer flasks and
placed on variable speed shaker for three days at room tmperature 26 ± 0.5 °C. The
seeds were treated with bacterial suspension by immersing them into the suspension
for 5 min. They were removed and planted on the same day. The inocula of the
pathogens were prepared by growing each fungus on autoclaved sorghum substrate
in 0.5 Kg milk bottles containing 20 g grains and 30 ml water, and autoclaved for 30
min at 20 lb/in2. After 4 weeks, the inoculum was mixed with the field soil at the
rate of 30 g inoculum per 1 Kg soil. The ratio was calculated on the basis of the
weight of the top 1 0 cm of field soil.
C. minitans was grown on sorghum substrate as previously described. The
peanut seeds were planted after one week of soil infestation. In the treatment of C.
minitans, teaspoon full of the inoculum was added to each hill immediately before
sowing.
To compare the efficacy of the biological treatment with chemical treatment,
Benomyl (Benlate 50 % W. P., Du Pont) carboxin Captan (Vitavax-Captan 75 % W.
P., Uniroyal), were used at the rate of 3 g/Kg seeds. All the treated seeds were
mixed with Rhizobium as recommended by the Department of Oil Crop, Agricultural
Research Centre, Egypt.
RESULTS AND DISCUSSION
Effect of P. lindbergii on fungal growth in PDA plates :
The growth of S. rolfsii was prevented opposite to the bacterial streak where
the mycelium became darkest. Also, F. oxysporum and R. so/ani growth was
406 K. K. SABET et al.
prevented for three days. All the tested fungi failed to grow in new PDA plates,
when 1 0 disks from edge opposite to the bacteria were transferred. This result
confirms findings of Lindberg (1981 ), who proved that P. lindbergii produced an
antibiotic lethal to a wide range of fungi including Fusarium and Rhizoctonia.
Effect of C. minitans on the tested fungi :
When 1 5 disks of each fungal treatment were transferred to new PDA, 9
of S. rolfsii, 6 disks of F. oxysporum and 5 disks of R. so/ani failed to produce
new growth. Presence of available nutrients in the medium has apparently
discouraged C. minitans to hyperparasitize other fungi; however, the ratio of failure
is not high. Results indicate that C. minitans could hyperparasitize the mycelium of
these fungi. Infection of the hyphae as well as sclerotia was confirmed by Huang and
Hoes (1976) and Trutmann and Keane (1982) when they studied its effect on S.
sclerotiorum.
The sclerotia of S. ro/fsii produced on sorghum substrate showed 55% failure
of germination, while those produced on PDA plates expressed 1 0 % failure of
germination compared with control showing ungerminated sclerotia of only 7.5 %.
This pronounced suppression of sclerotia! germination on sorghum substrate
suggested that they were attacked by C. minitans and could be one of its hosts
under laboratory conditions. Turner and Tribe ( 1 9 7 6) postulated that S. rolfsii
was not one of C. minitans hosts when they tested a Cyperus isolate from
carrots on PDA Universal bottle slopes. The same authors mentioned that not all
isolates of S. sclerotiorum were equally susceptible to C. minitans. Sclerotia
produced on PDA plates were less susceptible than those grown on autoclaved
carrot, probably because they matured in a drier atomsphere and became harder.
These results did not contradict ours, since we used a different isolate, and the
suscept ible sclerotia were those grown on sorghum which contained a relatively
high humidity.
The seed treatment with P. lindbergii alone was more effective than those
with C. minitans in reducing the pre- and post-emergence damping off (Table 1 ). lt
increased the percentage of plant survival by 50 % in the case of F. oxysporum and
R. so/ani and about 1 00 % in soil infested with S. rolfsii. Also, the treatment with
both P. lindbergii and C. minitans was highly effective but insignificantly less than
treatment with bacteria alone. Apparently, there might be a regative interaction
Tab
le 1
. E
ffe
ct o
f b
ioco
ntr
ola
nd
fu
ng
icid
al
tre
atm
ents
on
the
pe
rce
nta
ge
of
pre
-an
d p
ost
em
erge
nce
da
mp
ing
-o
ff.
Co
ntr
ol
ag
en
t a
P.
lind
be
rgii
1
C.
min
ita
ns
3
P.
lind
be
rgii
+ C
. m
init
an
s 2
Be
no
myl
1
8
Ca
rbo
xin
1
3
Non
e 3
a : p
erce
ntag
e o
f pr
e-e
mer
genc
e da
mpi
ng-o
ff.
b :
perc
enta
ge o
f pr
e-e
mer
genc
e da
mpi
ng-o
ff.
c :
perc
enta
ge o
f pl
ant
surv
ival
.
No-
Pat
hoge
n
b c
a
1 9
8
4
1 9
6
9
2 9
6
4
0 8
2
11
1 8
6
10
0 9
7
23 F.
oxy
spo
rum
R.
so/
ani
b c
a b
c a
2 9
4
6 1
93
8
6 8
5
13
16
71
18
4 9
2
7 3
90
10
6 8
3
12
9 7
9
10
3 8
7
7 5
88
10
13
64
2
6
13
61
33
5. r
olfs
ii
b 0 9 1 3 5 19
c 92
73
89
87
85
48
OJ 6 n 0 z -1
;;o 0 r v m
)>
z c -1
(/)
0 F
Tl c z <:!
:? ..p,.
0 "'-I
408 K. K. SABET et al.
between both antagonists.
Using of fu ngicides was moderately effective in protect ing the seedlings,
meanwhil e C. mini tans alone was the least effective. In th is respect, the latter
biocontrol agent didn't have enough t ime to hyperparasitize the pathogenes as the
hills were infested with it immediately before sowing. The same trend was observed
when the effect of these treatments on pod-rot was studied (Table 2), except that
the contro l effect of C. minitans appeared to be better than that on pre- and
post-emergance.
The fresh weight of plants from untreated seeds cultivated in infested soil
was less t han that of treated seeds in the same soil except where the soil was
Table 2. Effect of biocontrol and fung icidal treatments on the percentage of pod-rot.
% Pod-rot on 1 50 days peanut plants
Cont rol agent
No-Pathogen F. oxysporum R. so/ani 5. rolfsii
P. lindbergii 3.0 6.7 5.4 5.8
C. minitans 6.2 17.9 1 5.1 10.0
P. lindbergii +
C. minitans 3.5 7.1 6.7 7.4
Benomyl 1.0 14.0 15.0 13.0
Ca rbox in 0.0 9. 0 8.0 10.0
None 6.0 68.9 80.7 70 .2
BIOCONTROL - PEANUT - SOIL FUNGI 409
infested with 5. rolfsii (Table 3). The low plant density resulting from low
percentage of plant survival could play a role in this respect, as this pathogen alone
gave 48 % plant survival. The increase of bacterial nodules in treated plants was
attributed to the interaction between the pathogens and biocontrol agents. This
probability was supported by Knode et al. (1980) , who stated that, the reduction in
seed germination of blackgram ( Vigna mungo (L.) Wilczek), total number of nodules
and dry weight of plants were greater in inoculation with one fungus, Macrophomina
phaseoli, 5. rolfsii or F. oxysporum plus Rhizobium than with more than one fungus
and Rhizobium. They suggested that this effect might be due to the interaction
between the fungi. Also, Bandara (1978) argued that the presence of F. solani in
French bean rhizosphere soil reduced the number of root nodules probably following
interaction between the fungus and soil rhizobia rather than through direct effect on
the plant. The number of nodules in chemical treatment as seed dressing, were
generally lower than those of biocontrol treatment. This result confirmed that of
Ordish (1967) who mentioned that most of the seed dressers used to prevent
diseases of the plant also kill nodulating bacteria.
The treatment with P. lindbergii gave the highest protection as indicated by
seed weight and number per plant and the total seed yield of the planted row. The
treatment with C. minitans was less effective than the previous one, but still
significantly better than the untreated plants except in the case of 5. rolfsii. C.
minitans known to be a hyperparasite could be less aggressive with the former
fungus than with R. solani or F. oxysporum. On the other hand, it was effective in
protecting the pods from 5. rolfsii than from pre- and post-emergence damping-off.
This could be seen in Tables 1 and 2. In this respect, it might had long enough time
from date of sowing to pod formation to hyperparasitize the sclerotia or the
mycelium of the fungus. The fungicides showed some effectiveness in protecting the
seedlings or the pods but not as much as the bacteria did. Chemical treatment
affected the percentage of plant survival (Table 1) when it was used in uninfested
soil. This was in agreement with the findings of Zaher et al. ( 1984 ).
P. lindbergii treatment of uninfested soil showed high production in seed
number and weight per plant (Table 4) . Cook and Baker (1983) postulated that the
increased plant growth might result from the destruction of phytotoxins produced by
the rhizosphere microb iota or from inhibition of nonparasitic pathogenic
micro-organisms in the rhizosphere. The same authors argued that biological control
would be better than chemical control as seed dressing, since it lasts for a longer
Tab
le 3
. E
ffe
ct o
f b
ioco
ntr
ola
nd
fun
gici
dal
tre
atm
en
ts o
n fr
esh
& d
ry w
eig
ht
and
nu
mb
er
of
nodu
les
on p
lan
t ro
ots
.
No-
Pat
hoge
n ·
Co
ntr
ol a
gen
t a
P.
fin
db
erg
ii 2
81
0
C.
min
ita
ns
214
5
P.
lind
be
rgii
+ C
. m
init
an
s 26
86
Be
no
myl
13
00
Ca
rbo
xin
20
80
Non
e 2
66
0
Sx
105
.2
a :
aver
age
of
fres
h w
eig
ht
I 1
50
day
s ol
d pl
ant.
b :
aver
age
of
dry
we
igh
t I
1 5
0 d
ays
old
plan
t.
b
1335
1171
1236
80
0
1050
1500
170
c :
num
ber
of
nodu
les
form
ed o
n 1
50
day
s ol
d ro
ot
plan
t.
c
178.
4
186
.0
181.
4
121
.0
120
.1
178
28
F. o
xysp
oru
m
R. s
o/an
i
a b
c a
b c
a
14
30
5
10
178.
9 96
2 3
58
11
0.0
1330
945
30
8
79.8
9
36
34
9 10
8.1
1327
1392
49
9 13
9.8
84
8
355
102.
3 13
15
13
80
4
80
99
.8
882
309
98.1
89
0
1120
4
30
12
0.0
9
10
3
28
10
3.9
950
625
243
66.8
35
9 18
5 60
14
21
142
91
31
117
37
17.3
14
9 -
·-
-
5. r
olf
sii
b
508
41
0
500
325
305
452
48
c
137.
7
81.5
I I !
12
0.3
:
79.0
83.1
98.2
29.5
4:>-
......
0 ?"
?" ~ CI
J m
-;
~ ~
Tab
le 4
. E
ffe
ct o
f bi
ocon
trol
and
fung
icid
al t
rea
tme
nts
on
seed
num
ber
and
we
igh
t pe
r pl
ant
and
tota
l y
ield
per
ro
w.
Con
trol
age
nt
a
P.
lind
be
rgii
32
9
C. m
init
an
s 2
49
P. l
ind
be
rgii
+ C
. m
inita
ns
32
1
Ben
omyl
8
5
Car
boxi
n 1
48
.2
Non
e 3
72
Sx
63
a :
aver
age
num
ber
of
seed
s/ p
lant
.
b :
aver
age
we
igh
t o
f se
ed
s/p
lan
t, in
gra
ms.
c :
aver
age
we
igh
t o
f 1 0
0 s
eeds
in g
ram
s.
d :
tota
l yi
eld
/ro
w,
in g
ram
s.
No-
Pat
hoge
n
b c
d a
251.
3 7
6.4
5
02
6
23
2.2
161
64
.7
30
59
2
80
238.
1 7
4.2
4
52
4
28
8
83
.6
98
.4
13
38
14
0.8
17
9.8
1
21
.3
30
57
2
02
.1
25
5.7
6
8.5
5
114
100
69
.4
45
18
6
53
F. o
xysp
orum
R.
so/
ani
b c
d a
b c
261
93.1
5
151
170
.1
20
9.5
1
23.2
20
1.2
71.8
3
42
0
21
0
205.
1 9
7.7
22
7.0
7
8.8
4
08
6
20
1 2
07
.2
118
147
.9
105
25
14
1
50
.1
173
.6
11
5.7
20
3.1
10
0.5
3
45
3
14
7.5
1
64
.0
111.
2
116
.3
116
.3
151
2 1
48
1
35
.7
91.
7
37
32
.9
16
0
39
3
7.3
27
5.
rolfs
ii
d a
b c
39
81
29
7
20
3
68
.4
28
71
20
1 9
0.0
4
4.8
37
30
2
98
19
49
6
5.4
27
78
17
5 10
8.9
6
2.2
27
88
1
47
.5
18
9.1
1
28.
2
162
8
12
1 1
13
.3
93.
6
1 71
.4
43
167.
2 23
d
36
54
12
60
27
29
32
15
32
15
12
10
181.
7
"' 6 8 ~ (3
r -o r; z '=i (/
)
0 ;= ., c z e2
~
-'
J
412 K. K. SABET et al.
time and disperses around the rhi zosphere effectively without diluti on.
Furthermore, it is better from the economic and environmental points of view.
REFERENCES
1. Ahmed, A. H. M. and H. T. Tribe. 1977. Biological control of white rot of onion
Sclerotium cepivorum by Coniothyrium minitans. Plant Pathol., 26 : 75 - 78.
2. Bandara, J. M. R. S. 1978. Effect of Fusarium so/ani sp. Phaseoli on root
nodule formation in Phaseolus vulgaris L. Journal of the National Agric .
Society of Ceylon, 1 5 : 1 - 7.
3. Campbell, W. A. 1947. A new species of Coniothyrium parasitic on sclerotia.
Mycologia, 39 : 190 - 195.
4. Cook, R. J. and K. F. Baker. 1983. The Nature and Practice of Biological
Control of Plant Pathogens. The American Phytopathological Society, 539 pp.
5. Huang, H. C. and J. A. Hoes. 1976. Penetration and infection of Sclerotinia
sclerotiorum by Coniothyrium minitans. Can. J. Bot., 54 : 406 - 410.
6. Konde, B. K., D. B. Shinde and B. B. More. 1980. St udies on root infecting fungi
and Rhiz obium of blackgram ( Vigna mungo (L.) Wilczek). Journal of
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7. Lindberg, G. D. 1981. An antibiotic lethal to fungi. Plant Dis., 65 : 680 - 683.
8. Lindberg, G. D., H. A. Whaley and J. M. Larkin. 1980. Production of tropolone
by a Pseudomonas. J. Nat. Prod., 43 : 592 - 594.
9. Ordish, G. 1967. Pans Manual No. 2 pest Control in Groundnuts, 2nd edition.
Published in Britain by the Ministry of Overseas Development, 139 pp.
1 0. Sabet, K. K. 1987. Biological cont rol of corn stalk-rot by seed treatment with
bacterial suspension. Bull. Fac. Agric., Cairo Univ., 38 : 475 - 480.
11 . Tribe, H. T. 1957. On the parasitism of Sclerotinia trifoliorum by
Coniothyrium minitans. Trans. Br. Mycol. Soc., 40 : 489 - 499.
1 2. Trutmann, P., P. J. Keane and P. R. Merriman. 1980. Reduction of sclerotia!
inoculum of Sclerotinia sclerotiorum with Coniothyrium minitans. Soil Bioi.
Bochem., 12 : 461 - 465.
13. Trutmann, P. and P. J. Keane. 1982. Biological control of Sclerotinia
sclerotiorum on aerial parts of plants by the hyperparasite Coniothyrium
BIOCONTROL - PEANUT - SOIL FUNGI 413
minitans. Trans. Br. Mycol. Soc., 78 : 521 - 529.
14. Turner, G. J. and H. T. Tribe. 1976. On Coniothyrium minitans and its
parasitism of Sc/erotinia species. Trans. Br. Mycol. Soc., 66 : 97 - . 105.
15. Zaher, Effat A., A. A. EI-Shehidi, M. M. Satour and Ebtisam EI-Sherif. 1984.
Trichoderma harzianum Rifai, A. biocontrol agent effective against peanut
diseases. Bull. Fac. Agric., Cairo Univ., 35 : 545 - 561.
414 K: K. SABET et al.
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