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Mycopathologia et Mycologia applicata, vol. 44, 3 pag. 271-276, 197I INTERACTIONS OF ACTINOMYCETES WITH MACROPHOMINA PHASEOLI (MAUBL.) ASHBY; THE CAUSE OF ROOT ROT OF COTTON by ABDUL GHAFFAR 1) ABSTRACT Macrophomina phaseoli, the cause of root rot of cotton, was inhibited by Strepto- myees albus, S. griseus and S. noursei in agar culture. S. aureotaciens, S. flaveolus, S. rimosus, S. scabies and S. venezuelae were non-antagonistic. Only the antagonistic Streptomyces were found to reduce Macrophomina infection on cotton seedlings in soil without any deleterious effect on cotton growth. INTRODUCTION In a previous report (4) on the interactions of soil fungi with Macrophomina phaseoli, the cause of root rot of cotton, it was found that excepting Trichoderma viride and Penicillium nigricans, a majority of the fungal isolates tested did not have any adverse effect on M. phaseoli. Such similar observations were made in soil amended with organic substrates in which the control of M. phaseoli was re- lated to the increase in population of microorganisms antagonistic to M. phaseoli (5). Whereas the fungi were non-antagonistic, a number of bacteria and actinomycetes inhibited the growth of M. phaseoli in agar plate tests. Similarly the control of M. phaseoli infection on cotton seedlings was related to the increase in population of bacteria and actinomycetes in soil. In the present paper an account is given of the interactions of Streptomyces spp. with M. phaseoli in agar culture and in soil. MATERIAL AND METHODS 1. Isolation o/actinomycetes ]rom soil Samples of soil (sandy loam, pH 7.8) were obtained from a cotton field at the University of California, Riverside Farm near Moreno, California from which the actinomycetes were isolated by the soil dilution plate technique (13). One ml of 1 : 100,000 soil suspension was poured in sterilized Petri dishes and adequately dispersed with 1) Department of Botany, The University, Karachi, Pakistan. Accepted for publication: 18.IX.1970.

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Page 1: Interactions of actinomycetes with Macrophomina phaseoli (Maubl.) Ashby; The cause of root rot of cotton

Mycopathologia et Mycologia applicata, vol. 44, 3 pag. 271-276, 197I

I N T E R A C T I O N S OF A C T I N O M Y C E T E S WITH M A C R O P H O M I N A PHASEOLI (MAUBL.) ASHBY;

THE C A U S E OF ROOT ROT OF C O T T O N

b y

ABDUL GHAFFAR 1)

ABSTRACT

Macrophomina phaseoli, the cause of root rot of cotton, was inhibited by Strepto- myees albus, S. griseus and S. noursei in agar culture. S. aureotaciens, S. flaveolus, S. rimosus, S. scabies and S. venezuelae were non-antagonistic. Only the antagonistic Streptomyces were found to reduce Macrophomina infection on cotton seedlings in soil without any deleterious effect on cotton growth.

INTRODUCTION

In a previous repor t (4) on the in terac t ions of soil fungi wi th Macrophomina phaseoli, the cause of root ro t of co t ton , i t was found t h a t except ing Trichoderma viride a n d Penicill ium nigricans, a m a j o r i t y of the fungal isolates t es ted did not h a v e a n y adverse effect on M. phaseoli. Such s imilar observa t ions were m a d e in soil a m e n d e d wi th organic subs t r a t e s in which the control of M. phaseoli was re- la ted to the increase in popu la t ion of mic roorgan i sms an tagonis t ic to M. phaseoli (5). Whereas the fungi were non-antagonis t ic , a n u m b e r of bac te r i a a n d ac t inomyce te s inhib i ted the g rowth of M. phaseoli in agar p la te tests . Similar ly the contro l of M. phaseoli infect ion on co t ton seedlings was re la ted to the increase in popu la t ion of bac te r i a and ac t inomyce tes in soil. I n the present pape r an account is g iven of the in te rac t ions of Streptomyces spp. wi th M. phaseoli in agar cul ture a n d in soil.

MATERIAL AND METHODS

1. Isolation o/actinomycetes ]rom soil

Samples of soil ( sandy loam, p H 7.8) were ob t a ined f rom a co t ton field a t the U n i v e r s i t y of California, Rivers ide F a r m nea r Moreno, California f rom which the ac t inomyce tes were isolated b y the soil di lut ion p la te t echn ique (13). One ml of 1 : 100,000 soil suspension was poured in steri l ized Pe t r i dishes and adequa t e ly dispersed wi th

1) Department of Botany, The University, Karachi, Pakistan. Accepted for publication: 18.IX.1970.

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272 A. GHAFFAR

app' 10 ml of Czapek Dox agar, pH 7.2. Petri dishes were incubated at 25 ° C and actinomycetes growing in isolated colonies were trans- ferred on Czapek Dox agar slants. Amongst these Streptomyces albus and S. flaveolus were identified through the courtesy of Dr. T. G. PRIDItASI, Microbiologist, ARS, Northern Utilization Research & Development Division, Peoria, Illinois, 61604, USA.

2. Interaction in agar culture

The effect of 11 isolates of Streptomyces belonging to 8 different species on the growth of M. phaseoli was studied on Czapek Dox agar, pH 7.2. These included Streptomyces albus and S. flaveolus isolated from the soil as described above as well as S. griseus, S. scabies, S. aureo/aciens, S. noursei, S. rimosus, S. venezuelae from the culture collection of the Department of Plant Pathology, University of California, Riverside. The procedurein part was adap- ted as suggested by STESSEL et al. (11). Petri dishes containing about 10 ml of Czapek Dox agar, pH 7.2 was streaked on one side of the plate with a loopful of a suspension of actinomycetes in water. Inoculated plates were incubated at 25 ° C for 4 days after which 5 mm diam. disk of inoculum from the margin of an actively growing colony of M. phaseoli was placed on the plates at right angles to the streaks at a distance of about 55--60 rnm. The plates were again incubated at 30°C and the growth of M. phaseoli was recorded daily. Of the isolates tested only S. albus, S. griseus and S. noursei, known to produce antifungaI antibiotics (14), produced a zone of inhibition of 7--18 mm between the colonies of M. phaseoli and test organism (TaMe I, Fig. 1). With the remaining 5 species viz., S. aureo/aciens, S. flaveoh,,s, S. rimosus, S. scabies and S. venez~elae, known to produce generally antibacterial substances (14), M. phaseoli continued to grow uninhibited as in the control plates and

Fig. 1. In terac t ions of Actinomycetes wi th Macrophomina phaseoli on Czapek Dox agar after 4 days growth at 30 ° C. M. phaseoli on top of the dishes and the actino- mycetes on the bot tom. Left: M. phaseoli × Streptornyces flaveolus str. 3. (non-anta-

gonistic). Right: M. phaseoli × Streptomyces albus str. 2. (antagonistic).

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ROOT ROT OF COTTON '273

TABLE I

I n t e r a c t i o n of 11 isolates of Streptomyces spp. wi th Macrophomina phaseoli af ter 5 d a y s g r o w t h on Czapek D o x aga r a t 30 ° C

Tes t o r g a n i s m No. of Zone of Ant ib io t ics known to isolates inh ib i t ion be p roduced t e s t ed (mm) (V4aksman (14))

Streptomyces albus (RossI-DOR- 4 12 - -18 RIA) WAES~A~ & HENRICI

S. aureo/aciens DI~C~AR 1 0 S. /laveolus (WAKSMAN) WAKS- 1 0

MAN c~ I-IENEICI S. griseus ~rAKS?vIAN ~C HENRICI 1 8 S. noursei H~ZEN & BROWN 1 6 S. rimosus SOBIN, FINLAY ~; 1 0

KANE S. scabies (THAXTER) \¥AKSMAN 1 0

& HXNRICI S. venezuelae ]~HRLICH et al. I 0

Act inonlyce t in , Thio lu t in , E n d o m y c i n

Chloro te t racyc l ine A c t i n o m y c i n

S t r ep tomyc in , Cyc lohex imide N y s t a t i n Oxy te t r acyI in , R imoc id in

Ch lo ramphen ico l

touched the Streptomyces colonies in 4 days. I t may be mentioned that M. phaseoli has also been found to be inhibited by certain other actinomycetes viz., A ctinomyces griseus (10), Chainia antibiotica (12) and by antibiotics like Hygrostatin (2) and Hygroscopin (9) re- spectively produced by S. hygrostatius and S. hygroscopicus.

3. Antagonism in soil Following the screening for antagonistic properties of Strepto-

myces towards M. phaseoli, experiment was made to study the effect of S. albus, S. griseus, S. aureo/aciens and S. flaveolus on the develop- ment of M. phaseoli infection on cotton seedlings in soil. The soil used in this experiment was garden loam, pH 6.9 from the cotton field of the Botany Department, Karachi University. Cultures of Streptomyces spp. were grown for 10 days at 25 ° C in soil amended with 5 % cornmeal adjusted to 50 % w.h.c. (8) and sterilized in 100 g quantities in 250 ml flasks for 1 hr at 20 lb/in 2. These were later thoroughly mixed with unsterilized soil so as to give 1 : 4 and 1 : 1 parts by weight of inoculum and soil. Ordinary garden loam was used as control. The soils were transferred to 3.25" pots, 300 g per pot and 4 pots of each soil treatment. Two day old cotton seed- lings, var. M 100, dipped in a mycelial suspension of M. phaseoli (5) were transplanted into pots of soil, 5 seedling per pot. Noninoculated seedlings were transplanted in one pot of each treatment to see the phytotoxicity effects if any. After 2 weeks the seedlings were re- moved from the soil, washed in running water and examined under the microscope. Pathogenicity of M. phaseoli was determined by the presence of dark brown lesions on the roots and confirmed by making isolations from the root pieces on Czapek Dox agar. Of the 7 isolates

Page 4: Interactions of actinomycetes with Macrophomina phaseoli (Maubl.) Ashby; The cause of root rot of cotton

274 A. GHAFFAR

of Streptomyces tested, in the series where soil cultures of antgonists selected from agar plate tests were used, there was a reduction in the number of cotton seedlings infected with M. phaseoli especially at highest concentration of inoculum (Table II). Non-antagonistic

TABLE I I

Ef fec t of cu l tures of Slreptomyces spp. m i x e d in d i f ferent p ropor t ions wi th ga rden loam, on infect ion of co t ton seedl ings b y Macrophomina phaseoli, a t 50 % w.h.c .

T e s t o r g a n i s m No. of seedl ings in fec ted wi th 21/I. phaseoli :} I n o c u l n m : Soil 1 : 4 1 : 1

An tagon i s t i c in p la te cu l tu re Streptomyces albus Str. 1. Streptomyces albus Str, 2. Streptomyces albus Str. 4. Streptomyces Mbus Str, 5. Streptomyces griseus Str, T8 N o n - a n t a g o n i s t i c in p la te cu l tu re Streptomyces aureo]acienst Str. T485 Streptomyces flaveolus Str. 3 No an tagon i s t , control 20

20 16 16 14 20 15 20 18 16 12

20 20 20 20

:) O b s e r v a t i o n s based on 20 seedlings, 5 seedl ings per pot .

ones had no effect in the control of M. phaseoli infection. Similarly in the control series, where no antagonist was used, all the cotton seedlings were infected with M. phaseoli. It may be mentioned that none of the Streptomyces spp. had any deleterious effect on the growth of cotton seedlings.

C O N C L U S I O N

The present investigation of the interaction of actinomycetes with M. phaseoli on agar plate and in soil has given an indication that M. phaseoli is sensitive to Streptomyces spp. in its mycelial state. All the organisms which antagonized M. phaseoli in the agar plate reduced the incidence of root rot of cotton to a greater or lesser degree. Concentration of inoculum of these actinomycetes appeared to be an important factor. The importance of this inocu- lum potential as in the case of Trichoderne viride in the inactivation of rhizomorph production by Armillaria mellea is well known (3).

Various isolates of Streptomyces or their antibiotics have been found to reduce Pythium root rot in sugarcane (1) and damping off of alfalfa seedlings (6, 7). Assuming that the control of M. phaseoli was due to the production of antibiotics by antagonistic micro- organisms in soil, it would appear that the inoculum of Streptomyces added to the soil had sufficient concentration of antibiotics to reduce Macrophomina infection. The suppression of Macrophomina infection

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ROOT ROT OF COTTON 275

of cotton seedlings as reported earlier (5) could be attributed to a continuous production of antibiotics in soil by actinomycetes and bacteria in the presence of organic substrates and its subsequent decomposition by other microorganisms. Apparently the substrate, inoculum potential, etc., are all interrelated and need to be at opti- mum before control is accomplished. It would therefore be worth- while to examine this phenomenon in greater detail using organic amendments with addition of actinomycetes in soil.

Summary In t e r ac t i ons of 8 di f ferent species of Streptomyces with an isolate of Macrophomina

phaseoli f rom co t ton was s tud ied . Of these , Streptomyces albus, S. griseus and S noursei inh ib i ted t h e g r o w t h of IkI. phaseoli in aga r cu l ture . S. aureofaciens, S. /taveolus, S. rimosus, S. scabies a n d S. venezuelae, however , were n o n - a n t a g o n i s t i c a n d M. phaseoli was found to grow over t h e a c t i n o m y c e t e colonies.

Us ing a c t i n o m y c e t e s selected f rom aga r p la te tes ts , on ly t he an tagon i s t i c Strepto- myees were found to reduce M. phaseoli infect ion on co t ton seedl ings in soil. None of t h e a c t i n o m y c e t e s h a d a n y dele ter ious effects on co t t on growth .

Acknowledgement I wish to t h a n k Prof. D. C. ERWlN, D e p a r t m e n t of P l a n t Pa tho logy , U n i v e r s i t y of

California, Riverside for his encouragement and for critical review of the manuscript.

References 1. COOPER, W. E. ~: CHILTON, S. J. P. (1950) S tud ies on ant ib io t ic soil o rgan i sms .

i . A c t i n o m y c e t e s an t ib io t ic to Pythium arrhenomanes in suga rcane soils of Loui- s iana. Phytopathology, 40: 546--5522.

2. FURUSHIRO, K., SHIMIZU, K. , SA~]:AI, H., MtNOGATA, )/J'. & FUfflSAW&, T. (1960) t I y g r o s t a t i n , a new an t ib io t ic subs t ance . Chem. Abstr. 54: 10.

3. GARRETT, S. D. (1958) I n o c u l u m po ten t i a l as a fac tor l imi t ing le tha l ac t ion b y Trichoderma viride Ft . on Armillaria mellea (FR.) QUEL. Trans. Brit. Mycol. Soc. 41: 157--164.

4. GnAFFAR, A. (1968) I n t e r a c t i o n s of soil fung i wi th ~facrophomina phaseoli (M&UBL.) ASI~BY, t h e cause of roo t ro t of cot ton. ~lycopath. et Mycol. AppL 34: 196--201.

5. GHAFFAR, A., ZE~TMYER, G. A. & ERWlN, D. C. (1969) Ef fec t of organic a m e n d - m e n t s on seve r i ty of Macrophomina roo t rot of cot ton. -Phytopathology, 59: 1267--1269.

6. GREGORY, K. ~. , ALLEN, O. N., RIKER, A. J . (~ ~TERSON, ~ r H. (1952) Ant i - biot ics a n d 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 as control a g e n t s aga ins t d a m p i n g off of alfalfa. Phytopathology, 42: 613--622.

7. GREGORY, K. E., ALLEN, O. N., RIKER, A. J. & PETERSON, W. a. (1952) Anti- biotics as agents for the control of certain damping off fungi. Amer. J. Bot. 39: 405--415.

8. KEEN, ]3. A. & RACZKOWSKI, H. (192t) T h e re la t ion be tween t he c lay c o n t e n t a n d cer ta in phys ica l p roper t ies of a soil. jr Agric. Sci. 11: 441--449.

9. £-%~2xlqAZAWA, K., Oxi , K., TADOKORO, I., HONJO, iV[., HIT01KI, H. ~: UEYANGAGI, J. (1954) S tud ies on s t r e p t o m y c e t e s . Hygroscop in , an ant ib io t ic s u b s t a n c e aga ins t fung i a n d p h y t o p a t h o g e n s . J. Agric. Chem. Soc. Japan 28: 715--716.

10. ORSENIGO, M., ORSENIGO, LEDA t3. & ZUCCA RI]A (1955) An tagon i s t i c ac t ion of Actinomyces griseus UC 531 on va r ious fungi . Ann. Fac. Agri. Set. 2: 106--119.

11. STESSEL, G. J. , LEBEN CURT & KEITT, G. W ~. (1953) Screening t e s t s des igned to discover an t ib io t ics su i tab le for p l a n t disease control . Mycologia, 45: 325--334.

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276 A. GHAFFAR

12. THIRUMALACHAR, •. J. (1955) Chainia, a new genus of the Actinomycetales. Nature, Lond. 176: 934--935.

13. ~*VAKs~A~ ", S. A. (1927) Principles o5 soil microbiology. London: Bailliere Tyn- dall & Co.

14. VVAKSMAN, S. A. (1961) The Actinomycetes. VoI. II. Baltimore: The Williams & V¢ilkins Co.