PSEUDOMONAS LINDBERGH AND CONIOTHYRIUM MINITANS AS BIOCONTROL AGENTS EFFECTIVE AGAINST SOME SOIL...

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


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


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


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


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

Maharashtra Agriculture Universities, 5 : 222 - 225.

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


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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PSEUDOMONAS LINDBERGH AND CONIOTHYRIUM MINITANS AS BIOCONTROL AGENTS EFFECTIVE AGAINST SOME SOIL...

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