Suppression of Fusarium Wilt of Adzukibean by Rhizosphere

Chapter 29

Suppression of Fusarium Wilt of Adzukibean by Rhizosphere Microorganisms 1

2

2

Shinsaku Hasegawa , Norio Kondo , and Fujio Kodama 1

Hokkaido Institute of Public Health, North 19, West 12, North-ward, Sapporo 060, Japan The Hokkaido Central Agricultural Experiment Station, Naganuma 069-13, Japan

Downloaded by COLUMBIA UNIV on August 1, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch029

2

During a study on the biological contorol for Fusarium wilt of adzukibean caused by Fusarium oxysporum f. sp. adzukicola, we isolated antagonistic microorganisms from the rhizoshere soil of adzukibean root by the improved triple layer method. Hemipyocianine, chrororaphin and phenazine-l-carboxylic acid were isolated from Pseudomonas aeruginosa S-7 and P. fluorescens S-2; pyrrolnitrin from P. cepacia B-17; antibiotic Y-1 (monazomycin like) from Streptomyces flaveus Y-1. These microbial cultures and antifungal agents strongly inhibited growth of F. oxysporum f. sp. adzukicola FA-3. In greenhouse, the treatment of adzukibean seed with P. cepacia B-17 in F. oxysporum FA-3-infested soil decreased the diseases incidence from 76.3 to 8.8%; St. flaveus Y-1, 9.0%; P. aeruginosa S-7, 11.6%; P. fluorescens S-2, 13.3%. At a crop filed, the incidence decreased from 88% to 58%, 38%, 63% and 59% respectively. The isolates may be useful as antagonists to Fusarium wilt of adzukibean.

Fusarium w i l t of the adzukibean caused by Fusarium oxysporum f. sp. adzukicola i s a serious and widespred disease i n Hokkaido, northern part of Japan. This pathogen i s a root-infecting fungus that spreads r e l a t i v e l y slowly along the vascular bundle and causes necrosis, yellowing and w i l t ( l - 3 ) . In our work on Fusarium wilt of adzukibean, we have isolated microorganisms antagonistic to F. oxysporum f. sp. adzukicola FA-3 from the adzukibean rhizosphere, and report here: the i s o l a t i o n and i d e n t i f i c a t i o n of antagonists and their producing antifungal agents; and the efficacy of isoates or these antifungal agents as seed treatment to Fusarium w i l t by F. oxysporum. f . sp. adzukicola(l,4-6).

0O97-6156/91/0449-O4O7$06.00/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

408

NATURALLY OCCURRING PEST BIOREGULATORS

I s o l a t i o n and I d e n t i f i c a t i o n o f A n t a g o n i s t i c M i c r o o r g a n i s m s and t h e s e P r o d u c i n g A n t i f u n g a l Agents We i s o l a t e d a n t a g o n i s t s t o Fusarium oxysporum F A - 3 from the r i z o s phere o f a d z u k i b e a n and o t h e r m a t e r i a l s by t h e improved t r i p l e l a y e r

Table I. Isolated Antagonists and Their Producing Antifungal Agents

Pseudomonas aeruginosa S - l S-7 SH-6 f l u o r e s c e n s S-2 Y-15 NS-7371 c e p a c i a B-17 1218

Adzukibean(Rl) Adzukibean(Rl) Soil Adzukibean(Rl) Melone(Rl) liiy(Rl) Adzukibean(Rh) Beet(Rh)

Streptomyces sp. No. 2 sp. B-6 flaveus Y - l

Adzukibean(Rl) Adzukibean(Rl) R i v e r water

N

0

>luteorii

Source( )*1

Lororaph:

Hemipyocianinc


Antagonist

1 -H 0) i H

rrolnitrj

i

a Antifungal agents

+

+ -

e + + + (+)

(+)*2 + + + + +

-

-

+

+ +

Water is o l u b l e Water is o l u b l e Antibiotic Y - l (monazomycin l i k e )

*1 Sample s o u r c e : ( R l ) , R h i z o p l a n e ; (Rh), Rhizosphere *2 A n t i f u n g a l agent: p r o d u c i n g , +; t r a c e , (+); no p r o d u c i n g , Hemipyocianine

|

H

}j

Pyrrolnitrin

CONH

2

Chlororaphin Pyoluteorin

Phenazine-1carboxylic acid

^1 OH

COOH

CI

O

HO

H

9H OH HO

Antibiotic Y - l (Monazomycin like)

HO OH

OH

OH OH

•CH3 OH

O H ^ H

CH3 C H g C H 3 OH >H 8H H

CH

HN

v

OH

OH

2

CH3 CH3

CH3 CH~

CH

3

CH

3

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

29.

HASEGAWA ET AL.

Suppression of Fusarium Wilt of Adzukibean 2

409

5

method(4). One ml of diluted(10 -10 w/v) rhizosphere s o i l of adzukibean were spread i n plates with 1.5% agar and added the spore suspension of F. oxysporum FA-3 on the plates. These plates were incubated at 28 C for 2 to 4 days, and then the colonies with clear zone were picked up. These i s o l a t e s were i d e n t i f i e d as Pseudomonas aeruginosa, P. fluorescens, P. cepacia, Streptomyces flaveus and Streptomyces spp.( 1,2,4-8, Table I). These produced hemipyocianine, chlororaphin, phenazine-l-carboxylic a c i d ( l , 6 ) , p y r r o l n i t r i n ( 1 , 4 ) , pyoluteorin(5,6), A n t i b i o t i c Y-l(monazomycin l i k e , 7) and water soluble unknowns(Table I ) .


in vitro Antifungal A c t i v i t i e s of Microbial Cultures and A n t i fungal Agents against Fusarium oxysporum f. sp. adzukicola FA-3 Bioassay by reversed layer method(4, F i g . I) showed that 3 s t r a i n s of Pseudomonas and St. flaveus Y-l(8) were highly i n h i b i t o r y to F. oxysporum FA-3(Table II). On agar spot inoculation plates(4, F i g . I ) , P. aeruginosa S-7, St. flaveus Y-l and Streptomyces sp. No.2 showed highly i n h i b i t i o n and continuance of antifungal a c t i v i t i e s . The minimum i n h i b i t o r y concentration(MIC) of p y r r o l n i t r i n against F. oxysporum FA-3 was 3.13 îg/ml; A n t i b i o t i c Y - l , 6.25 pg/ml; hemipyocianine, 50 pg/ml; chlororaphin and phenazine-lcarboxylic acid, 100 ug/ml; and water soluble unknowns, 25-50 jug/ml (Table I I ) . Antifungal Spectrum of Microbial Cultures and Antifungal Agents The microbial cultures of i s o l a t e s and t h e i r antifungal agents strongly inhibited growth of F. oxysporum and also many other plant pathogenic fungi: F. solani, F. moniliforme, F. roseum, Biporaris sorokiniana, Alternaria alternata, Cladosporium cucumerinum, Pyricularia oryzae, Pythium graminicolum, Verticillium dahliae, Cylindrocarpon sp., Rhizoctonia solani(Table I I I , IV).

a. Reversed layer method Fig. I

b. Agar spot i n o c u l a t i o n method

Antifungal a c t i v i t i e s of antagonists against Fusarium oxysporum FA-3



410

A n t i b i o t i c Y - l was an e f f e c t i v e i n h i b i t o r of a l l tested pathogens i n c u l t u r e . But p y r r o l n i t r i n was much l e s s e f f e c t i v e against Fusarium species than any other genera, and was t o t a l l y i n e f f e c t i v e s against F. oxysporum f.sp. cepae. Also, phenazines, such as hemipyocianine did not i n h i b i t strongly the growth of Fusarium species(Table IV). But the s t r a i n s which produced phenazines were a c t i v e against Fusarium species and continued these strength on the agar spot i n o c u l a t i o n method(Table I I I ) . The s p e c i f i c i t y of the s t r a i n s f o r s u s c e p t i b i l i t y against antifungal agents were recognized.


Table I I The I n h i b i t i o n e f f e c t of i s o l a t e d antagonists and t h e i r a n t i fungal agents against Fusasrium oxysporum FA-3 Antagonist

I. Antagonist agar d i s c

I I . Antifungal agent

Reversed layer method*!, 2days (inhibitioni zone, mm)

Isolated antifungal agent

Pseudomonas aeruginosa S-7 (Brucella agar, 30 'C, 4days)

30.0

Pseudomonas cepacia B-17 (Brucella agar, 30 *C. 4days)

33.8

Pseudomonas fluorescens NS-7371 (Brucella agar, 30 *C. 4days)

31.5

Streptomyces sp. No. 2 (YM agar*3, 30*C, 7days)

24.0

Streptomyces sp. B-6 (PDA*4, 30 'C, 4days)

25.0

Streptomyces flaveus Y - l (GS agar*5, 30 C, 7days)

31.2

Agar spot inoculation method*2 (inhibition zone, mm)

9.5 +++(7days)

Hemipyocyanine Chlororaphin Phenazine-lcarboxylic a c i d

7.0 -H- (4days)

pyrrolnitrin

9.5 +++(4days)

3.5 +

3.13

Pyrrolnitrin Phenazine-lcarboxylic a c i d

3.13 200

(7days)

11.2 -H-K4days)

Crude water soluble

25

Crude water soluble

50

9.5 +++(7days) 4.7 ++ (4days) 0.0 -

(7days)

10.7 +++(4days)

{

#

100 200 200

(7days)

7.2 ++ (4days) 4.8 +

Minimum inhibitory concentration (ug/ml, 7days)

Antibiotic Y-l (Monazomycin like)

3.13

10.1 +++(7days)

*1 Reversed layer method: Fusarium oxvsprum FA-3 spores were used f o r bottom layer and 4 o r 7 days c u l t u r e agar d i s c s of antagonists were used. *2 Agar spot i n o c u l a t i o n method: 7 days culture agar d i s c s of Fusarium oxysporum FA-3 and 4 or 7 days culture agar discs of antagonists were used. I n h i b i t i o n zone between the pathogen and the antagonist: +++£ 9.0mm, $.0>-H->5.0, 5.0>+;>2.0, 2.0>(+)>0.0, — 0 . 0 *3 YM agar: Yeast-malt agar *4 PDA: Potato dextrose agar *5 GS agar: Glycerol-soybean meal agar


In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. +++ +++

+++ +++

++ ++ +++ +++ +++

++ ++ ++

+++ ++

-

++ ++ ++ ++ + +++ +++ +++ +++ +++

-

7

+++ +++

+

-

++

++ + + + ++

++ +

+++ +++

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++f +4+ +4+

++ ++ ++ ++ + ++

+4+ + + +

++

++ ++ ++ +++ +++ ++ ++ ++ +++ +++ +++ ++ +++ +++

4 ++ ++

+ +

7

4

4

3 O (V 3 I MHW PH 4-1 Z

+++ +++*5++ ++ ++ ++

7

Pu O CO

(0 V I fr, (0 to

3 (0

»iH O 0)

i

C 0)

«J c

(0 (0

7

++ ++ +++ ++ ++ + + + ++ +++ +++ ++ 4-4-4+4-4+4-+ ++

+++ +++ -H-f +++ ++ + + + ++ +++ +++ ++ +++ +++

+++ +++ +4+ +++

4

r 4J O

4

ND*6++

-

7

++ ++ ++4h +++

++ +

+

++ +-

+

++ +

7

+++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ 4h++ +++ NT* 7

4h++ 4h++ 4h++ 4h++ 4h4+ 4h++ 4-++ 4h++ 4-++ 4h++ 4h++ 4h++ 4h++ 4h++

Hh++ +++ 4h++ ++

4

0) > H (0

a 9.0mm, 9.0>++>5.0, 5.0>+>2.0, 2.0> (+)>0.0. — 0 . 0 ~ ~ *6 ND: not detected. *7 NT: not tested.

Fusarium oxysporum FA-3(adzukibean)*3 Fusarium oxysporum FGH(adzukibean) Fusarium oxysporum F-l(adzukibean) Fusarium oxysporum f.sp.*4 spinacea H-18 Fusarium oxysporum f.sp.spinacea H-29 Fusarium oxysporum H-32(green pepper) Fusarium oxysporum f . s p . f r a g a r i a e H-34 Fusarium oxysporum f.sp.melonis H-34 Fusarium oxysporum f . s p . l y c o p e r s i c i KF-244 Fusarium oxysporum f.sp.cepae KF-228 Fusarium oxysporum KF 8 5 4 ( l i l y ) Fusarium s o l a n i H-41(rice) Fusarium moniliforme H-24(rice) Fusarium moniliforme H-36(rice) Fusarium roseum H-35(wheat) B i p o r a r i s sorokiniana H-21(rice) A l t e m a r i a a l t e m a t a H-25(rice) Cladosporium cucumerinum H-23(melon) P y r i c u l a r i a oryzae H-22(rice) Pythium graminicolum H-30(rice) V e r t i c i l l u m d a h l i a e H-28(strawberry) Cylindrocarpon sp. KF 8 4 6 ( l i l y ) Rhizoctonia s o l a n i H-31(potato) Rhizoctonia s o l a n i KF 8 5 2 ( l i l y )

Culture days

O -H

09 C M

«J

Antagonists

Growth i n h i b i t i o n o f the plant pathogens by the m i c r o b i a l c u l t u r e s o f a n t a g o n i s t s * !

Phytopathgenic fungi*2

Table I I I


412



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29.

HASEGAWA ET A L .

Supression of Fusarium Wilt of Adzukibean

E f f i c a c y of Seed Bacterization with M i c r o b i a l of Fusarium Wilt of Adzukibean

413

Cultures to Control

In greenhouse, treatment of the adzukibean seed with washed b a c t e r i a l c e l l s of P, cepacia B-17 i n F. oxysporum FA-3-infested s o i l ( F i g . I I ) decreased the disease incidence from 76.3 to 8.8% (Fig. I l l ) ; S t . flaveus Y - l , 9.0%; P. aeruginosa S-7, 11.6%;

Adzukibean


ANTAGONIST

BACTERIZATION

NO TREATMENT

/

Dipping Incubation Fig. I I B i o l o g i c a l control of Fusarium w i l t

F i g . I l l E f f e c t o f seed b a c t e r i z a t i o n with Pseudomonas cepacia B-17 on the c o n t r o l of Fusarium w i l t o f adzukibean caused by Fusarium oxysporum FA-3*1 *1 Adzukibean: Hayate-syouzu, Treatment: Pathogenic f u n g i , Fusarium oxysporum FA-3(10 spores/g Antagonist, Pseudomonas cepacia B-17(10 cells/ml s o l u t i o n ) . 5

8


soil);

414


Table V

E f f e c t s of seed b a c t e r i z a t i o n with antagonists on the control of adzukibean w i l t caused by Fusarium oxysporum i n green house and crop f i e l d * ! Disease incidence(Z) Green house (36days)


Antagonist*2 Pseudomonas aeruginosa S-7 Pseudomonas cepacia B-17 Pseudomonas fluorescens NS-7371 Flavobacterium sp. AB7 Acinetobacter calcoaceticus B28 Streptomyces sp. No. 2 Streptomyces sp. B6 Streptomyces flaveus Y - l Without antagonist

Crop f i e l d (45days)

-

-

11.6 8.8 20.5 30.2 77.0 15.2 65.0 9.0 76.3

63 58 -*3

-

85 92 82 50 88

Me-cellulose 60 46

41-

-

38 88

*1 Adzuibean: Hayate-shozu i n green house; Takara-shozu i n crop field, Pathogenic fungi: Fusarium oxysporum FA-3, 10 spores/g s o i l i n green house; Fusarium oxysporum 10 c~ells/g s o i l i n crop f i e l d *2 Antagonist: 108cel?.s/ml s o l u t i o n *3 Not tested. 5

3

Streptomyces sp. No.2, 15.2%; and P. fluorescens NS-7371, 20.5% (Table V). In crop f i l e d , treatment with St. flaveus Y-l cultures decreased the disease incidence from 88 to 38%; Streptomyces sp. No.2, 41%; and P. cepacia B-17, 46%(Table V). Methyl c e l l u l o s e was used for i n j e c t i o n of microbial c e l l s at the time of seeding i n field. Growth of Microorganisms and Production of Antifungal Agents i n S o i l on the Control of Fusarium Wilt of Adzukibean In the case of low disease incidence, such as P. cepacia B-17 and St. flaveus Y - l , the increase of the number of antagonistic microorganisms and the decrease of the number of pathogen, F. oxysporum FA-3, i n the rhizosphere s o i l of adzukibean were recognized(Fig.IV, Table VI). The t i g h t l y relationship between the increase of a n t i fungal agents and the decrease of disease incidence was not recognized. Washed microbial c e l l s were as efficacious as whole cultures or culture f i l t r a t e s , even though no or a few amount of antifungal agents were present when the seeds were treated. None of the treatments was phytotoxic to adzukibean. The protective effect exhibited by treatment of adzukibean seed with cultures of the antagonistic microorganisms may be due to release of the antifungal agents by the gradual growth of the microbial c e l l s . Even though the concentration may be low, the lysing c e l l s may e f f e c t prolonged release and a v a i l a b i l i t y of the antifungal agents during the c r i t i c a l period of seeding growth.


29.

HASEGAWA ET AL.

Pseudomonas cepacia 10

415

Suppression of Fusarium Wilt of Adzukibean

B-17

Streptomyces f l a v e u s Y - l

c

Without antagonist 10

;

3 8l0 ° »

w2

4

3 10 3

IS


10 20

10

30

Culture days

F i g . IV

20

30

Culture days

Time course comparison of growth of microorganisms and production of a n t i f u n g a l agents on the c o n t r o l of Fusarium w i l t of adzukibean i n green house

Table VI Growth of microorganisms and production of antifungal agents i n rizoshere s o i l of adzukibean i n green house Number of c e l l s / g

soil

Antagonist Antagonist*l (days)O 10 20 30 36 Pseudomonas aeruginosa S-7

F. oxysporum*2 0 10 20 30 36 T

Production of antifungal agent (ug/g s o i l ) 0 10 20 30 36

Disease incidence

T~

Pseudomonas cepacia B-17 Pseudomonas fluorescens NS-7371 Acinetobacter calcoaceticus B-28 Streptomyces sp. No. 2

*1 10®cells/ml solutions of antagonists were used f o r seed bacterization. *2 10 c e l l s / g s o i l of Fusarium oxysporum FA-3 were used.



416

Conclusion These r e s u l t s suggested that the i s o l a t e s such as P. cepacia B-17, St. flaveus Y-l and Streptomyces sp. No.2 may be useful as antagonists to F. oxysporum f . sp. adzukicola and may f a c i l i t a t e establishment of c u l t i v a t i o n of healthy adzukibean. The antagonisms exhibited by the microorganisms are possibly the r e s u l t of production of antifungal agents, which are themselves e f f e c t i v e i n protecting agaist Fusarium w i l t of adzukibean. Literature Cited


1. 2. 3. 4. 5.

6. 7. 8.

Hasegawa, S.; Kodama, F.; Kaneshima, H . ; Akai, J. J . Pharmacobio-Dyn. 1987, 10S, 57. Kodama, F.; Hasegawa, S. Kongetu no Nouyaku 1985, 30, 22-27 Kondo, N . ; Kodama, F. Shokubutu Boeki 1989, 43, 28-33. Hasegawa, S.; Kamneshima, H . ; Kodama, F.; Akai, J. Report of the Hokkaido Institute of Public Health 1986, 36, 16-23. Hasegawa, S.; Kondo, N . ; Kodama, F . Proceedings of 7th Symposium on the Development and Application of Naturally Occurring Drug Materials, 1989, 7, 67-70. Hasegawa, S.; Kondo, N . ; Kodama, F. J . Pharmacobio-Dyn. 1990, in press. Hasegawa, S.; Kamneshima, H. Report of the Hokkaido Institute of Public Health 1987, 37, 6-17. Hasegawa, S.; Kodama, F.; Nakajima, M . ; Akai, J.; Murooka, H. B u l l . Agr. & Vet. Med., Nihon Univ. 1987, 44, 80-86.

RECEIVED May 16, 1990


Suppression of Fusarium Wilt of Adzukibean by Rhizosphere

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