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

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<ul><li><p>Mycopathologia et Mycologia applicata, vol. 44, 3 pag. 271-276, 197I </p><p>INTERACTIONS OF ACTINOMYCETES WITH MACROPHOMINA PHASEOLI (MAUBL.) ASHBY; </p><p>THE CAUSE OF ROOT ROT OF COTTON </p><p>by </p><p>ABDUL GHAFFAR 1) </p><p>ABSTRACT </p><p>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. </p><p>INTRODUCTION </p><p>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 major i ty 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 populat ion of microorganisms antagonist ic to M. phaseoli (5). Whereas the fungi were non-antagonist ic, a number of bacter ia and act inomycetes 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 populat ion of bacter ia and act inomycetes 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. </p><p>MATERIAL AND METHODS </p><p>1. Isolation o/actinomycetes ]rom soil </p><p>Samples of soil (sandy loam, pH 7.8) were obtained from a cotton field at the Univers i ty of California, Riverside Farm near Moreno, California from which the act inomycetes were isolated by the soil di lution plate technique (13). One ml of 1 : 100,000 soil suspension was poured in steril ized Petr i dishes and adequately dispersed with </p><p>1) Department of Botany, The University, Karachi, Pakistan. Accepted for publication: 18.IX.1970. </p></li><li><p>272 A. GHAFFAR </p><p>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 &amp; Development Division, Peoria, Illinois, 61604, USA. </p><p>2. Interaction in agar culture The effect of 11 isolates of Streptomyces belonging to 8 different </p><p>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 30C 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 </p><p>Fig. 1. Interactions of Actinomycetes with 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 bottom. Left: M. phaseoli Streptornyces flaveolus str. 3. (non-anta- </p><p>gonistic). Right: M. phaseoli Streptomyces albus str. 2. (antagonistic). </p></li><li><p>ROOT ROT OF COTTON '273 </p><p>TABLE I </p><p>Interact ion of 11 isolates of Streptomyces spp. with Macrophomina phaseoli after 5 days growth on Czapek Dox agar at 30 C </p><p>Test organism No. of Zone of Antibiot ics known to isolates inhibit ion be produced tested (mm) (V4aksman (14)) </p><p>Streptomyces albus (RossI-DOR- 4 12--18 RIA) WAES~A~ &amp; HENRICI </p><p>S. aureo/aciens DI~C~AR 1 0 S. /laveolus (WAKSMAN) WAKS- 1 0 </p><p>MAN c~ I-IENEICI S. griseus ~rAKS?vIAN ~C HENRICI 1 8 S. noursei H~ZEN &amp; BROWN 1 6 S. rimosus SOBIN, FINLAY ~; 1 0 </p><p>KANE S. scabies (THAXTER) \AKSMAN 1 0 </p><p>&amp; HXNRICI S. venezuelae ]~HRLICH et al. I 0 </p><p>Actinonlycetin, Thiolutin, Endomyc in </p><p>Chlorotetracycl ine Act inomycin </p><p>Streptomycin, Cycloheximide Nystat in OxytetracyI in, Rimocidin </p><p>Chloramphenicol </p><p>touched the Streptomyces colonies in 4 days. It 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. </p><p>3. Antagonism in soil Following the screening for antagonistic properties of Strepto- </p><p>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 </p></li><li><p>274 A. GHAFFAR </p><p>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 </p><p>TABLE I I </p><p>Ef fect of cultures of Slreptomyces spp. mixed in different proport ions with garden loam, on infection of cotton seedlings by Macrophomina phaseoli, at 50 % w.h.c. </p><p>Test organism No. of seedlings infected with 21/I. phaseoli :} Inocu lnm: Soil 1 : 4 1 : 1 </p><p>Antagonist ic in plate culture Streptomyces albus Str. 1. Streptomyces albus Str, 2. Streptomyces albus Str. 4. Streptomyces Mbus Str, 5. Streptomyces griseus Str, T8 Non-antagonist ic in plate culture Streptomyces aureo]acienst Str. T485 Streptomyces flaveolus Str. 3 No antagonist , control 20 </p><p>20 16 16 14 20 15 20 18 16 12 </p><p>20 20 20 20 </p><p>:) Observat ions based on 20 seedlings, 5 seedlings per pot. </p><p>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. </p><p>CONCLUSION </p><p>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). </p><p>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 </p></li><li><p>ROOT ROT OF COTTON 275 </p><p>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. </p><p>Summary Interact ions of 8 different species of Streptomyces with an isolate of Macrophomina </p><p>phaseoli f rom cotton was studied. Of these, Streptomyces albus, S. griseus and S noursei inhibited the growth of IkI. phaseoli in agar culture. S. aureofaciens, S. /taveolus, S. rimosus, S. scabies and S. venezuelae, however, were non-antagonist ic and M. phaseoli was found to grow over the act inomycete colonies. </p><p>Using act inomycetes selected from agar plate tests, only the antagonist ic Strepto- myees were found to reduce M. phaseoli infection on cotton seedlings in soil. None of the act inomycetes had any deleterious effects on cotton growth. </p><p>Acknowledgement I wish to thank Prof. D. C. ERWlN, Depar tment of P lant Pathology, Univers i ty of </p><p>California, Riverside for his encouragement and for critical review of the manuscript. </p><p>References 1. COOPER, W. E. ~: CHILTON, S. J. P. (1950) Studies on antibiotic soil organisms. </p><p>i. Act inomycetes antibiotic to Pythium arrhenomanes in sugarcane soils of Loui- siana. Phytopathology, 40: 546--5522. </p><p>2. FURUSHIRO, K., SHIMIZU, K., SA~]:AI, H., MtNOGATA, )/J'. &amp; FUfflSAW&amp;, T. (1960) t Iygrostat in , a new antibiot ic substance. Chem. Abstr. 54: 10. </p><p>3. GARRETT, S. D. (1958) Inocu lum potent ial as a factor l imit ing lethal act ion by Trichoderma viride Ft. on Armillaria mellea (FR.) QUEL. Trans. Brit. Mycol. Soc. 41: 157--164. </p><p>4. GnAFFAR, A. (1968) Interact ions of soil fungi with ~facrophomina phaseoli (M&amp;UBL.) ASI~BY, the cause of root rot of cotton. ~lycopath. et Mycol. AppL 34: 196--201. </p><p>5. GHAFFAR, A., ZE~TMYER, G. A. &amp; ERWlN, D. C. (1969) Effect of organic amend- ments on sever i ty of Macrophomina root rot of cotton. -Phytopathology, 59: 1267--1269. </p><p>6. GREGORY, K. ~., ALLEN, O. N., RIKER, A. J. (~ ~TERSON, ~r H. (1952) Anti - biotics and antagonist ic micro-organisms as control agents against damping off of alfalfa. Phytopathology, 42: 613--622. </p><p>7. GREGORY, K. E., ALLEN, O. N., R IKER, A. J. &amp; PETERSON, W. a. (1952) Anti- biotics as agents for the control of certain damping off fungi. Amer . J. Bot. 39: 405--415. </p><p>8. KEEN, ]3. A. &amp; RACZKOWSKI, H. (192t) The relation between the clay content and certain physical propert ies of a soil. jr Agric. Sci. 11: 441--449. </p><p>9. -%~2xlqAZAWA, K., Oxi, K., TADOKORO, I., HONJO, iV[., HIT01KI, H. ~: UEYANGAGI, J. (1954) Studies on streptomycetes. Hygroscopin, an antibiotic substance against fungi and phytopathogens . J. Agric. Chem. Soc. Japan 28: 715--716. </p><p>10. ORSENIGO, M., ORSENIGO, LEDA t3. &amp; ZUCCA RI]A (1955) Antagonist ic act ion of Actinomyces griseus UC 531 on var ious fungi. Ann. Fac. Agri. Set. 2: 106--119. </p><p>11. STESSEL, G. J., LEBEN CURT &amp; KEITT, G. W ~. (1953) Screening tests designed to discover antibiot ics suitable for p lant disease control. Mycologia, 45: 325--334. </p></li><li><p>276 A. GHAFFAR </p><p>12. THIRUMALACHAR, . J. (1955) Chainia, a new genus of the Actinomycetales. Nature, Lond. 176: 934--935. </p><p>13. ~*VAKs~A~ ", S. A. (1927) Principles o5 soil microbiology. London: Bailliere Tyn- dall &amp; Co. </p><p>14. VVAKSMAN, S. A. (1961) The Actinomycetes. VoI. II. Baltimore: The Williams &amp; Vilkins Co. </p></li></ul>

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