1 HI I l 4 l‘ W W H! H H| 1| \I w } l " .__. - _ k_ ._ 7; _'___,_ m [M \ { l 5; \h‘ MECHANISM OF ENHIBITWN 0F FUNGI EN AGAR BY ACTENOMYCETES Thesis for the Degree of M: S. MECHEGAN. STATE UNSVERSITY SU‘CHAN HSU 1968 LIBRARY “1i ‘ Michigan State University THESIS ‘ 3:: Biplane BY ‘7’ ‘ ~ HUN 5 SUNS' § .l N am mm mg. IIIIIIIIIIII ABSTRACT MECHANISM OF INHIBITION OF FUNGI IN AGAR BY ACTINOMYCETES by Su-Chan Hsu Experiments were designed to study the nature of the inhibition of fungi in agar by actinomycetes. Specif— ically. the possibility was tested that inhibition. in some cases. might be due to nutrient competition rather than due to antibiotics. Twenty unidentified actinomycete isolates were tested for antagonism to Glomerella cingulata and Mucor ramannianus in agar media. All 20 actinomycetes inhibited .fl. ramannianus. and 18 inhibited g. cingulata. Some inhi- bition zones produced by the actinomycetes were shown to contain inhibitory substances. Agar disks from inhibition zones. or paper disks placed beneath inhibition zones. of 9 actinomycete isolates made new inhibition zones when transferred to fresh. seeded agar. By the same methods. antibiotics could not be demonstrated in inhibition zones of the other ll isolates. Further. antibiotics were 1 Su—Chan Hsu produced in liquid cultures by 8 of the same 9 isolates which produced them in agar media. and no antibiotics were detected in liquid cultures of the other ll isolates. Conidia of g. cingulata. which do not require exo— genous nutrients for germination. germinated in cultures of non-antibiotic producing actinomycetes but failed to germinate in cultures of antibiotic—producing actinomycetes. Sizes of inhibition zones produced by non-antibiotic pro- ducing actinomycetes against g. ramannianus. which needs exogenous nutrients for germination. were decreased when the concentration of nutrients in the medium was increased. However. zone sizes produced by antibiotic-producing actino- mycetes did not decrease when the medium contained increas- ing concentration of nutrients. When agar media containing glucose (0.2%) and glu- tamic acid (0.2%) were streaked with 4 isolates of actino- mycetes. these compounds were rapidly utilized adjacent to the actinomycete colonies. About 80% of the glucose and glutamic acid were lost from the agar near the actinomycete colonies after 6 days incubation. Conidia of g. ramannianus germinated about 80% in a solution of glucose and glutamic 2 Su-Chan Hsu acid equal to that used in the agar medium. and germinated about 45% in 20% of the original concentration of nutrients. Agar was leached by allowing sterilized distilled water to run slowly for 7 days through a groove cut in the agar. A clear inhibition zone developed on either side of the groove when conidia of g. ramannianus or g. cingulata were sprayed on the agar surface and water was again allowed to run through the groove for 3 more days. More than 90% of the glucose and amino acids had been leached from the agar. These results indicate that while some antagonistic actinomycetes inhibit develOpment of fungi in agar by pro- duction of antibiotics. others do so by means of nutrient competition. MECHANISM OF INHIBITION OF FUNGI IN AGAR BY ACTINOMYCETES by Su-Chan Hsu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1968 \f‘t) ACKNOWLEDGMENTS Deep appreciation is expressed to Dr. J. L. Lockwood for his guidance and encouragement throughout this study. and Special thanks to him for assistance during the course of this investigation and in preparation of the manuscript. Thanks are also due to Dr. J. E. Cantlon. Dr. M. L. Lacy and Dr. R. P. Scheffer for their critical evaluation of the manuscript. I would also like to extend my appreciation to Mr. P. G. Coleman for photographs. ii TABLE OF CONTENTS ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . . . . . . . . . . . MATERIALS AND METHODS. . . . . . . . . . . . . . . . . Preparation of actinomycetes and fungi . . . . . . Assays of inhibition zones . . . . . . . . . . . . Assays for lysis . . . . . . . . . . . . . . . Detection of antibiotics . . . . . . . . . . . . Utilization of nutrients in agar by actinomycetes. RESULTS. . . . . . . . . . . . . . . . . . . . . . . Inhibition characteristics of actinomycetes. Effect of pH on inhibition zones . . . . . . . . . Lysis characteristics of actinomycetes . . . . Antibiotic production in liquid media. . . . Relation of antibiotic production to inhibition of fungal spore germination in actinomycete cultures iii Page ii vi 10 12 12 18 20 23 23 Table of Contents——cont. Page Effect of nutrient enrichment on inhibition zones. 27 Nutrient status of agar adjacent to actinomycete colonies . . . . . . . . . . . . . . . . . . . . 30 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 37 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 41 iv LIST OF TABLES Table Page 1. Inhibition characteristics of actinomycetes against Glomerella cingulata in agar tests . . l3 2. Inhibition characteristics of actinomycetes against Mucor ramannianus in agar tests. . . . l4 3. Effect on pH on inhibition zones of Glomerella cingulata. . . . . . . . . . . . . . . . . . . l9 4. Lysis of Glomerella cingulata by actinomycetes in agar. . . . . . . . . . . . . . . . . . . . 21 5. Production of antibiotics by actinomycetes in linquid media. . . . . . . . . . . . . . . . . 24 LIST OF FIGURES Figure 1. Inhibition characteristics of actinomycetes against Glomerella cingulata . . . . . . . . . Germination of Glomerella cingulata conidia in actinomycete cultures with and without added potato broth . . . . . . . . . . . . . . . . . Effect of concentration of glucose and peptone on size on inhibition zones produced by anti— biotic and non-antibiotic actinomycetes. . . . Effect on nutrient concentration on size of inhibition zones produced by actinomycetes . . Loss of glucose from 7 mm diam. agar disks immediately adjacent to actinomycetes. . . . . Loss of glutamic acid from 7 mm diam. agar disks immediately adjacent to actinomycetes. . . . . DevelOpment of inhibition zone on leached agar Vi Page 15 26 28 29 31 32 36 MECHANISM OF INHIBITION OF FUNGI IN AGAR BY ACTINOMYCETES INTRODUCTION Many actinomycetes can produce antibiotics which inhibit fungi in artificial media. Some inhibitory micro- organisms do not appear to produce antibiotics, but until now experimental evidence implicating factors other than antibiotics was not available to explain inhibition in agar media. The widespread fungistasis occurring in natural soil has recently been interpreted as due to a deficiency of nutrients required by fungal spores for germination in the soil (9, 10, 11). K0 and Lockwood (9) showed that failure of fungal spores to germinate on agar discs incu— bated on soil was correlated with rapid loss of nutrients from the agar discs. Moreover, agar disks could be made fungistatic by leaching them with sterile water. This sug- gested the possibility that the inhibition zones produced by microorganisms in agar might, in some cases. be due to 1 depletion of nutrients adjacent to the antagonistic colony. The purpose of this study was to investigate the mechanism of the inhibition of fungi by actinomycetes in agar, and particularly to determine if nutrient depletion might be a mechanism of antagonism. LITERATURE REVIEW When several microbes are growing in the same cul- ture medium, growth of some will be repressed in proximity to other microbes, whereas others will continue to grow. This is usually attributed to the production by some orga- nisms of inhibitory substances which diffuse through the medium and repress to growth of others at some distance away (2, 3, 4, 5, 25). Porter (18) in an early paper speculated that antagonisms displayed by fungi in mixed cultures are due either to exhaustion of nutrients or to the formation of detrimental products. Florey £5 31. (5) indicated that an organism which shows antagonism on solid media will often not produce any detectable inhibitory sub— stances in a liquid medium containing the same nutrient materials. He speculated that in some cases this was due to the lack of ample aeration in liquid cultures, but in other cases the reason for the phenomenon was obscure. Some other suggestions for the mechanism of microbial antag- onism include change in the pH reaction in the medium and enzyme action (4, 5, 17, 18, 23). 3 In numerous agar platings of mixed microbial popu- lations from natural substrates, the antagonism of fungi by actinomycetes, rather than by fungi, has been more fre- quently observed (4, 20, 23), but not all actinomycetes can inhibit fungi. In one study, 43 to 51% of the actinomy- cetes tested by the agar streak method were active against one or more fungi. However, heated culture filtrates from the antagonistic cultures, when tested by the diffusion procedure, showed that only 43%.had inhibitory properties (20, 24). Stessel gt §l° (21) studied the occurrence in soil of actinomycetes active against plant pathogenic fungi. 0f 21 isolates of actinomycetes inhibitory to glgmr erella cingulata, only 16 produced an antibiotic substances in the liquid media. Nakhimovskaia (16) also found that of 47 isolates of actinomycetes possessing antagonistic prOp— erties in agar media only 27 secreted antibiotic substances into the medium. Meredith (13) in a survey of the distri- bution of organisms antagonistic to Fusarium oxysporum cubense in Jamaican soil, found 17 actinomycetes antago- nistic to this fungus. Of these only 8 produced inhibitory substances in the medium. Inhibition of fungi in liquid media may also occur without the presence of demonstrable antibiotic substances. No antibiotics could be demonstrated when washed cells of bacteria or Streptomyces spp. inhibited the germination of spores of test fungi in buffer solutions or in water in the absence of added nutrients (10). In several studies, when antagonistic cultures were tested for the presence of antibiotics using paper or thin layer chromatography, some cultures did not show the pres- ence of antibiotics (7, 8, 28). Many reports have indicated that the composition of the culture medium is important to the production of antibiotics by antagonistic microorganisms. The formula- tions of some of these media have been summarized by Waks- man and others (3, 4, 19, 23). Many of these media have contained materials of natural origin, such as soybean meal and other seed meals (1), peptone and other digests (2). Despite much effort in developing these media, many antag- onistic organisms still failed to produce antibiotics. Recent information suggests that failure of fungal spore germination in soil is due to depletion of nutrients by microbial activity rather than to the presence of inhibitory substances (9, 10, 11). The inhibition of spore germination on agar placed in contact with soil may be caused by the diffusion of nutrients from the agar rather than by the diffusion of fungistatic materials into agar. Agar disks supplemented with glucose or amino acids showed a rapid loss of these nutrients to soil by diffusion. Moreover, agar disks could be made fungistatic by leaching them with sterile water (9, ll). MATERIALS AND METHODS Preparation of actinomycetes and fungi.—-Twenty uni— dentified actinomycete isolates from soil were maintained on potato-dextrose agar. Glomerella cingulata (Ston.) Spauld. & Schrenk and Mucor ramannianus Mbller were used as test fungi; Conidia of g. cingulata do not require exogenous nu- trients. whereas conidia of M. ramannianus require exogenous nutrients for germination. Both were maintained on potato- dextrose agar. Conidia were obtained by washing the surface of the agar slants with sterilized distilled water. Assays of inhibition zones.--Tests for inhibitory activity were made in petri dishes on peptone agar (per liter: peptone. 2 or 5 g; agar. 20 g) or peptone—glucose agar (per liter: peptone. 2 or 5 9; glucose. 2 or 5 g; agar. 20 g). Actinomycetes were streaked on the media. and incubated at 24 C for 2 or 3 days. Then spore suspensions of test fungi. g, cingulata or M. ramannianus. were sprayed onto the agar surface with a modified Stansly's Spray apparatus (8). Approx- imately 250.000 spores were Sprayed onto each plate. After 3 days. the inhibition zones were measured. The presence of antibiotics in the inhibition zones was tested by 3 methods: (a) Agar disks, 7 mm in diam., were cut from inhibition zones and transferred to peptone agar or peptone—glucose agar, or to petri dishes without agar, and allowed to incubate without supplying additional inoculum of the test fungus. Presence or absence of growth of the test fungus was observed; (b) Agar disks, 7 mm in diam., were cut from inhibition zones and transferred to peptone agar or peptone—glucose agar. Immediately or sev- eral hours later, a spore suspension of the same test fun- gus was sprayed onto the agar surface. The presence or absence of new inhibition zones was observed; (c) Sterile paper disks, 13 mm in diam., were placed beneath the agar of the inhibition zones and kept for 10-15 days to allow any inhibitory substances to diffuse into the paper disks. The disks were then transferred to peptone agar or peptone- glucose agar, and the test fungus was sprayed on the agar surface. The develOpment of any new inhibition zones was observed. Assays for lysis.—-Actinomycetes were asSayed for ability to lyse the mycelia of g, cingulata. Mycelia were prepared by mixing a suspension containing approximately 20,000 conidia per ml of water with warm (42 C) peptone agar at the rate of 1 volume of spore suSpension to 5 vol— umes of agar. The agar was then poured in petri dishes and incubated for 2-3 days. Actinomycete isolates were then streaked on the surface of the agar. At 10, 25, and 30 days, the lytic zones were measured and the lysed my— celia was observed under the microsc0pe. After 30 days, agar disks 7 mm in diam. were cut from lytic zones and transferred to peptone or peptone—glucose agar. A spore suspension of g, cingulata was sprayed onto the surface of the agar to test for the presence of diffusible inhib- itory substances. Other disks were transferred to peptone agar containing growing mycelium of g. cingulata to test for diffusible lytic substances. Detection of antibiotics.--Actinomycetes were cul- tured in 125 ml Erlenmyer flasks containing 20 m1 liquid medium per flask. The media were (a) Difco—Bacto nutrient broth, 6 9; glucose, 5 9; NaCl, 5 g; ZnSO4 7H20, 0.01 g: CaC03, 3.5 g; distilled water, 1000 ml. (b) Peptone, 5 g: glucose, 5 g; distilled water 1000 m1. Actinomycete iso— lates were grown, in duplicate, in these shaken media for 3 days at 24 C. Sterilized Millipore filters (0.22 n or 10 0.44 H) were used to obtain sterile culture filtrates. Five ml of each filtrate were applied to 13 mm. diam. paper disks by applying 0.1 ml at a time then alternately air- drying the disks. Assays for antibiotic activity were car— ried out by the agar diffusion method (5, 15). The paper disks impregnated with culture filtrates were placed on the surface of 0.5% peptone agar. The test fungus, g. cingulata, was then sprayed on the agar surface. The as- say plates were incubated at 24 C for 3 days, when the zones of inhibition were observed. Utilization of nutrients in agar by actinomycetes. --An agar medium was prepared in 0.1 M phosphate buffer (pH 7) and contained glucose, 2 g; glutamic acid, 2 g; agar, 25 g; distilled water, 1000 ml. This medium supported good growth of the test fungi and actinomycetes. Two layers of agar were prepared. The bottom layer of 5 ml was poured and distributed smoothly and evenly in the petri plate. Four selected isolates of actinomycetes were streaked on the agar surface. On the lst, 2nd, 4th, and 6th days, 6 agar disks, 7 mm in diam., from the top layer of agar near the actinomycete colonies were removed and melted in 3 ml glass-distilled water. Glucose was assayed using the 11 Glucostat reagent (Worthington Biochemical Corporation). The reagent was used according to the manufacturer's di- rections. The tubes were read in a colorimeter at 400 mu. Glucose at concentrations of 20, 40 and 80 u/ml was used as a standard. Glutamic acid was assayed by melting 3 disks in 6 ml glass—distilled water, using the method of Moore and Stein (l4). Glutamic acid at concentrations of 4, 8 and 16 ug/ml was used as a standard. RESULTS Inhibition characteristics of actinomycetes.—-To determine which actinomycetes were capable of producing inhibition zones in agar, 20 unidentified actinomycete isolates were streaked on peptone—glucose agar or peptone agar using Glomerella cingulata or Mucor ramannianus as the test fungus. Each isolate was tested in duplicate in each experiment, and at least 3 experiments were done. Of the 20 actinomycetes, 18 made inhibition zones (3-19 mm) against gt cingulata (Table l) and 20 made inhibition zones (7—19 mm) against M. ramannianus (Table 2). To determine whether the agar in the inhibition zones contained antibiotics, additional tests were made. For g. cingulata, agar disks from the original inhibition zones of 9 isolates (1A, 2A, 4A, 6A, 7A, 10A, 16A, 24A and 28A) made new inhibition zones when transferred to fresh agar plates seeded with g, cingulata (Table 1, Fig. l). New inhibition zones were also produced by these isolates when paper disks placed beneath the original inhibition zones were transferred to fresh seeded agar. When agar 12 13 TABLE 1. Inhibition characteristics of actinomycetes against Glomerella cingulata in agar tests Inhibition characteristics of agar from inhibition zones Actinomycete Inhibition New inhibition Growth of test isolates zone (mm)a zones (mm) fungus Agarb ::§::c Agard Glasse 1A 18 8 9 — - 2A 18 8 9 - — 3A 13 0 0 + + 4A 18 9 10 - - 5A 0 0 6A 15 10 10 — — 7A 18 6 4 - - 8A 15 0 0 + + 9A 3 O O + + 10A 19 10 10 — - 12A 4 0 0 + + 13A 4 0 0 + + 15A 3 0 0 + + 16A 12 5 7 - - 17A, 13 0 0 + + 18A 4 0 0 + + 19A 10 0 0 + + 24A 15 8 8 - - 26A 0 0 28A l9 13 10 - - a . Actinomycetes were streaked on the agar. After 3 days. conidia of the test fungus were sprayed on the agar surface. b . . . . . Agar disks from inhibition zones were transferred to agar subsequently sprayed with conidia of g, cingulata. cPaper disks were placed beneath the inhibition zones. then transferred to agar seeded with g. cingulata. dAgar disks from inhibition zones were transferred to un— seeded agar; -=No growth of test fungus. +=Growth of test fungus. eAgar disks from inhibition zones were transferred to petri dishes without agar; -=No growth of test fungus. +=Growth of test fungus. 14 TABLE 2. Inhibition characteristics of actinomyctes against Mucor ramannianus in agar tests Inhibition characteristics of agar from inhibition zones Actinomycete Inhibition New inhibition Growth of test isolates zone (mm)a zones (mm) fungus Agarb :::::c Agard Glasse 1A 19 7 7 - - 2A 18 7 8 - — 3A 16 O O + + 4A 19 8 8 — 5A 7 O 0 + + 6A 18 7 6 - - 7A 14 7 5 - - 8A 16 O O + + 9A 7 O O + + 10A 19 10 8 - — 12A 7 O 0 + + 13A 8 O O + + 15A 7 0 O + + 16A 18 5 4 - - 17A 13 0 0 + + 18A 11 O O + + 19A 13 O O + + 24A 14 9 6 - - 26A 8 O O + + 28A 17 7 10 - - a . Actinomycetes were streaked on the agar. After 3 days. conidia of the test fungus were sprayed on the agar surface. bAgar disks from inhibition zones were transferred to agar subsequently Sprayed with conidia of M. ramannianus. CPaper disks were placed beneath the inhibition zones. then transferred to agar seeded with M, ramannianus. dAgar disks from inhibition zones were transferred to un- seeded agar; -=No growth of test fungus. +=Growth of test fungus. eAgar disks from inhibition zones were transferred to petri dishes without agar; -=No growth of test fungus. +=Growth of test fungus. Fig. 1. Inhibition characteristics of actino— mycetes against Glomerella cingulata. Above—left: The top 2 streaks. 1A and 4A.produced antibiotics. the bottom 2 streaks. 3A and 8A,were nonantibiotic—producing actino— mycetes. Above-right: Paper disks placed beneath the corresponding original inhibition zones were transferred to fresh seeded agar. Below—left: agar disks from cor— reSponding inhibition zones were transferred to unseeded agar. Below—right: Agar disks from corresponding inhi— bition zones were transferred to seeded agar. 16 disks from the original inhibition zones were transferred to fresh agar or petri dishes without agar, the test fungus failed to grow from disks of the same 9 isolates. These results showed that diffusible inhibitory substances were produced by 9 of the actinomycetes. Agar disks from zones produced by the other 9 an- tagonistic isolates (3A, 8A, 9A, 12A, 13A, 15A, 17A, 18A and 19A) did not make new inhibition zones when transferred to new agar plates seeded with g, cingulata. Nor were new inhibition zones produced by these 9 isolates when paper disks placed beneath the original inhibition zones were transferred to fresh seeded agar. Agar from zones produced by these isolates supported growth of the test fungus when disks were transferred to fresh agar or to petri dishes with- out agar. These results indicated that no diffusible in- hibitory substances were produced by these actinomycetes and that the inhibition zones were caused by some other factor. The size of inhibition zones containing inhibitory substances ranged from 12—19 mm, with a mean of 17 mm. The size of inhibition zones without inhibitory substances ranged from 3-15 mm, with a mean of 8 mm. 17 In the case of M. rammannianus, agar disks or paper disks from inhibition zones of 9 isolates (1A, 2A, 4A, 6A, 7A, 10A, 16A, 24A and 28A) made new inhibition zones, when transferred to new agar plates seeded with M. ramannianus (Table 2). The test fungus did not grow on the agar disks from inhibition zones of the same 9 isolates transferred to fresh agar: the same results were obtained even when a drop of 0.2% of glucose solution was added to the agar disks. The results are the same as those with g. cingulata and indicated further that the actinomycetes produced anti- biotics in the medium. Agar disks or paper disks from inhibition zones of the other 11 isolates (3A, 5A, 8A, 9A, 12A, 13A, 15A, 17A, 18A, 19A and 26A) did not produce new inhibition zones when transferred to new agar plates seeded with M. ramannianus. Agar disks from the original inhibition zones of the same 11 isolaes supported growth of the test fungus when disks were placed on fresh agar in petri dishes, or in a petri dish without agar when a drop of glucose solution was added. From these tests it was concluded that the actinomycete iso- lates included two kinds of antagonistic cultures, one pro- ducing inhibitory substances, and the other not producing inhibitory substances. 18 The size of inhibition zones containing inhibitory substances ranged from 14—19 mm, the mean being 17 mm. The size of inhibition zones without inhibitory substances ranged from 7-16 mm with a mean of 11 mm. Effect of pH on inhibition zones.-—To determine if pH changes in the agar could account for the inhibition zones of non—antibiotic producing isolates, the 20 actino— mycete isolates were streaked on unbuffered peptone agar (pH 6.9) and on peptone agar containing 0.1 M phosphate buffer (pH 7) and incubated for 5 days. A spore suspen- sion of the test fungus, g, cingulata, was then sprayed on the plates. After 3 days, the sizes of the inhibition zones, pH of the agar in zones and pH of the agar outside of the zones (growth area) were measured (Table 3). In peptone agar either with or without buffer, the pH of the inhibi- tion zones and the corresponding growth area did not differ appreciably. The pH of agar without buffer increased from 6.9 to 8.5 both in inhibition zones and in the growth area. Since g, cingulata grew well even at pH 8.5, the inhibition zones could not be attributed to an unfavorable, elevated pH of the medium. This was confirmed in tests with buffered agar, in which the pH remained essentially unchanged both 19 TABLE 3. Effect on pH on inhibition zones of Glomerella cingulata No Buffer. pH 6.9 0.1 M PO Buffer. pHT7 Actinomycete a a :22: .H :22: .H (mm) Zone GrOth (mm) Zone Growtg area ' area 1A 17 7.2 8.0 15 6.9 6.9 2A 15 8.2 8.0 14 6.9 6.9 3A 17 8.1 8.0 17 6.9 6.9 4A 18 8.1 8.0 17 6.9 6.9 5A 0 8.4 0 6.8 6A 17 8.4 8.5 17 6.9 6.9 7A 16 7.8 8.0 13 6.9 6.9 8A 14 7.6 8.0 10 6.9 6.9 9A 3 7.6 7.5 2 7.0 7.0 10A 19 7.8 7.9 16 7.0 7.0 12A 5 8.2 8.2 3 7.0 7.0 13A 5 8.1 8.2 3 7.0 7.0 15A 2 8.3 8.3 2 6.9 6.9 16A 16 8.1 8.1 l 6.9 6.9 17A 17 8.2 8.1 1 7.0 7.0 18A 3 7.9 8.0 3 7.1 7.1 19A 13 7.9 8.0 l 7.1 7.1 24A 19 8.1 8.1 16 7.1 7.1 26A 0 8.1 0 6.9 28A 19 8.2 8.1 13 6.9 6.9 apH was measured by placing electrodes on the agar sur- face after the cultures had grown for five days. bArea outside of the inhibition zones where the test fungus grew. 20 in the inhibition zones and in the growth area. Zone sizes for the same isolates were similar in both buffered and unbuffered agar with the exception of isolates 16A, 17A and 19A, which produced much smaller zones in the buffered agar. Similar small zones were produced by isolates 16A, 17A and 19A in agar buffered with 0.1 or 0.01 M phosphate buffer at either pH 7.0 or 8.0. In all tests, these 3 isolates grew poorly on buffered agar, whereas they grew normally on un- buffered agar. Therefore, the aberrant results with these isolates appear to be due to poor growth of the actinomycetes on buffered agar rather than to any effect of pH. Lysis characteristics of actinomycetes.--The 20 actinomycete isolates were tested for their ability to lyse ‘g. cingulata mycelium. Thirteen isolates (1A, 2A, 3A, 4A, 6A, 7A, 8A, 10A, 16A, 17A, 19A, 24A and 28A) produced lytic zones after 10 days incubation (Table 4). The size of lytic zones ranged from 3-7 mm and the mycelium in these zones was less dense when examined with the microsc0pe. After 25 days, the size of lytic zones produced by the 13 isolates increased and 3 additional isolates (9A, 12A and 13A) made small lytic zones. After 30 days, 2 additional actinomycetes made lytic zones. The mycelium in the lytic zones produced 21 TABLE 4. Lysis of Glomerella cingulata by actinomycetes in agar , Zone diameter (mm) Effect of transferred Actino- Degree lysis zone mycete of . . . isolates 10 25 3O lysisa , Inhlbltlon days days days LySis zone (mm) 1A 3 12 14 + O 8 2A 6 12 15 + 0 8 3A 6 6 8 + 0 0 4A 6 7 10 + 0 6 5A 0 O O — O 0 6A 4 8 13 + O 7 7A 5 12 15 + O 3 8A 7 8 12 ++ O 0 9A 0 3 4 + O 0 10A 5 12 15 ++ O 8 12A 0 2 6 + O 0 13A 0 3 8 + 0 0 15A 0 0 0 - 0 0 16A 3 7 10 + 0 6 17A 3 7 8 + O 0 18A 0 O 2 + O 0 19A 4 9 12 + O 0 24A 6 6 12 + O 6 26A 0 0 2 + 0 0 28A 8 9 15 + O 8 a — = No lysis of mycelium. + = Partial lysis. ++ = Com- plete lysis. 22 by 16 isolates (1A. 2A. 3A. 4A. 6A. 7A. 9A. 12A. 13A. 16A. 17A. 18A. 19A. 24A. 26A. and 28A) was partially lysed. whereas mycelia in the zones produced by isolates 8A and 10A were completely lysed. Agar disks from lytic zones of these 18 actinomycetes were transferred to fresh test fungal cultures. but none made new lytic zones. Agar disks from lytic zones were also tested for the presence of antibi- otics by transferring them to fresh. seeded peptone agar. Once again. the same 9 isolates (1A. 2A. 4A. 6A. 7A. 10A. 16A. 24A. and 28A) which produced transferable inhibition zones also made such zones when disks from lytic zones were transferred to new. seeded agar. However. the other 9 isolates (3A. 8A. 9A. 12A. 13A. 17A. 18A. 19A. and 26A) which caused lysis of g. cingulata mycelium demonstrated no antibiotics in the agar when tested this way. Therefore. the lytic zones produced by these 9 isolates must be caused by factors other than antibiotics or other diffusable lytic factors. At 30 days. the sizes of lytic zones containing antibiotics ranged from 10-15 mm. with the average. 12 mm. The sizes of lytic zones without antibiotics ranged from 2—12 mm. and averaged 7 mm. 23 Antibiotic production in liquid media.-—To further test whether antibiotics were produced only by the previously designated actinomycete isolates. antibiotic production was tested in 2 kinds of liquid media. Sixteen isolates of ac- tinomycetes. 9 of which produced diffusible inhibitory sub- stances in agar and 7 of which did not produce inhibitory substances. were tested. Sterile culture filtrates of 8 isolates (1A. 2A. 4A. 6A. 10A. 16A. 24A. and 28A) contained antibiotic substances (Table 5). Filtrates of the other 8 isolates (3A. 5A. 7A. 8A. 9A. 12A. 13A. and 18A) contained no detectable inhibitory substances. The 8 isolates pro- ducing antibiotics in liquid media were the same as those producing transferable inhibition zones on agar. whereas with one exception the other isolates did not produce trans— ferable inhibition zones on agar plates. This isolate. 7A. grew poorly in the liquid media. and this may account for its failure to produce antibiotics in these tests. Thus. with this exception. the results confirm those of tests done in agar. Relation of antibiotic production to inhibitign of fungal spore germination in actinomycete cultures.--To fur- ther investigate the inhibitory cultures that were unable 24 TABLE 5. Production of antibiotics by actinomycetes in liquid media Actinomycete Medium Aa Medium Bb isolates 1A +C + 2A + + 3A — - 4A + + 5A — 6A + + 7A — - 8A - — 9A — - 10A + + 12A - — 13A — - 16A + + 18A - — 24A + + 28A + + Control - - aNutrient broth 69. glucose 5g. NaCl 5g. Zn SO4 7H20 0.01g. CaCO 3.5g. distilled watter 1000 ml. 3 bPeptone 5g. glucose 5g. distilled water 1000 ml. C+ = Production of antibiotics; — = no production of antibiotics. 25 to produce inhibitory substances. 4 isolates of actinomy— cetes were selected. Isolates 1A and 4A produced inhibi— tory substances. and 3A and 8A did not produce detectable inhibitory substances. Conidia of g. cingulata. which do not require exogenous nutrients for germination. were in- cubated directly in 3-day-old cultures of these actinomy— cetes grown in liquid media. Conidia of g. cingulata were introduced directly into the actinomycete cultures and in- cubated for 14 hours. when germination was determined. A low level of germination. 11% and 18%. occurred in cultures of isolates 1A and 4A. respectively (Fig. 1). These re- sults were not significantly altered when potato broth was added at the time conidia were introduced. This confirmed that these cultures produce some inhibitory substances. By contrast. a high level of germination. 77% and 72%. occurred in cultures of 3A and 8A. respectively. When potato broth was added to these cultures. 98% of g. cingulata conidia germinated. indicating that actinomycetes cultures 3A and 8A did not contain any inhibitory substances (Fig. 2). The results are consistent with the view that no inhibitory sub- stances inhibitory to the test fungi are produced by actino— mycete isolates 3A and 8A. 26 100 GERMINATION. % 0| 0 TA 4A 3A 8A CK TA 4A 3A 8A CK NO POTATO BROTH POTATO BROTH Fig. 2. Germination of Glomerella cingulata conidia in actinomycete cultures with and without added potato broth. Isolates 1A and 4A producrd in— hibitory substances; isolates 3A and 8A did not pro- duce inhibitory substances. Data are the mean values of three experiments. In each experiment 150—180 Spores were counted in each of 10 microscopic fields. 27 Effect of nutrient enrichment on inhibition zones.-- If isolates such as 3A and 8A produce inhibition zones as results of exhaustion of nutrients from the agar surround— ing the actinomycete colonies. agar containing increasing amounts of nutrients should give inhibition zones decreas— ing in size. To test this possibility. 9 different concen— trations (0.2-4.0%) of peptone and glucose were prepared in agar. The 4 representative isolates (1A. 3A. 4A. and 8A) of actinomycetes were streaked on the agar media and incubated for 3 days. Conidia of the test fungus. M. ramanneanus. a species requiring nutrients for germination. were sprayed on the agar surface. After 3 days. inhibition zones were measured. The size of inhibition zones produced by isolates 3A and 8A gradually decreased as the concentration of nutri- ents increased in the medium (Fig. 3. 4). Agar disks from these inhibition zones produced no new inhibition zones when transferred to fresh. seeded agar. and the test fungus grew when similar agar disks were transferred to unseeded agar. The size of inhibition zones produced by isolates 1A and 4A did not decrease as the concentration of nutrients increased in the medium. Agar disks from these inhibition zones pro- duced new inhibition zones when transferred to fresh seeded '5 1 I f V r I I V 10 E E m‘ 6 N Z 9 ': m - 5 3: Z O I 1 l l l L l I 0 I 2 3 4 CONCENTRATION OF NJTRIENTS, °/o Fig. 3. Effect of concentration of glucose and peptone on size of inhibition zones produced by antibiotic and non-antibiotic actinomycetes. Actino— mycetes 1A and 4A were antibiotic producers. 3A and 8A were non—antibiotic producers. 29 Fig. 4. Effect of nutrient concentration on size of inhibition zones produced by actinomycetes. The concen- tration of peptone and glucose increased from above—left to below-right. The top 2 isolates in each petri dish were antibiotic-producing actinomycetes (1A and 4A); the bottom 2 isolates in each petri dish were non-antibiotic— producing actinomycetes (3A and 8A). 3O agar. and the test fungus did not grow when similar agar disks were transferred to unseeded agar. The same results were obtained in two additional tests. Therefore. these results provide further evidence that inhibition zones pro— duced by isolates 3A and 8A were caused by depletion of nutrients in the agar. Nutrient status of agar adjacent to actinomycete ‘colonies.--Several lines of evidence indicated that some of the actinomycetes inhibited fungi by some means other than antibiotic production. probably depriving the medium of nu- trients. Therefore. the nutrient status of agar near the actinomycete colonies was investigated. To determine the rate at which nutrients were utilized by actinomycetes from agar. glucose (0.2%) and glutamic acid (0.2%) were used as the sole ingredients in the agar. Actinomycete isolates 1A. 3A. 4A. and 8A and the test fungus. M. ramannianus. grew well on this medium. Glucose was rapidly lost from the agar within 7 mm of all of the actinomycete streaks; 60-70% was lost by the second day. and 80~90% was lost by the 6th day (Fig. 5). Glutamic acid was also rapidly utilized by the 4 actinomycetes. More than three—fourths of the glutamic acid was lost by the 6th day (Fig. 6). AS before. agar 31 2000 g . . j . 1 _ CONTROL) a: EISOOr < E .. ‘~ U! a. ”-1000- 0'! O u . :3 8 500- O l 1 1 1 i 0 I 2 3 4 5 6 DAYS Fig. 5. Loss of glucose from 7 mm diam. agar disks immediately adjacent to actinomycetes. Agar containing glucose 0.2% and glutamic acid 0.2% was used. Actinomycetes 1A and 4A were antibiotic producers; 3A and 8A were non—antibiotic producers. In the control. no actinomycetes were streaked on the agar. 32 2000 ;_7 j ' _ CONTROL) IN q d 1500 L I 1000 500 T GLUTAMIC ACID, p9/ml AGAR Fig. 6. Loss of glutamic acid from 7 mm diam. agar disks immediately adjacent to actinomycetes. Agar containing glucose 0.2% and glutamic acid 0.2% was used. Actinomycetes 1A and 4A were antibiotic producers; 3A and 8A were non-antibiotic producers. In the control. no actinomycetes were streaked on the agar. 33 disks from the area adjacent to isolates 1A or 4A. when transferred to fresh seeded agar. made new inhibition zones. but agar disks from the area adjacent to isolate 3A or 8A. when transferred to fresh seeded agar. did not make new inhibition zones. Experiments were done to determine the effect of. amount of glucose and glutamic acid on germination of con— idia of the test fungus. M. ramannianus. Two levels of each compound were used. a "low" amount (250 ug/ml of each compound). and a "high" level (2000 ug/ml). The low level approximates the amount of each compound remaining in the agar after the 5th day. whereas the high level correSponds to the amount originally in the medium. The compounds were prepared in the following mineral salt solution: NaNO . 3 2 9; K HPO . l g; MgSO 7H 0. 0.5 9; KCl. 0.5 g; FeSO 7H 0. 2 4 4 2 4 2 0.01 g: water. 1000 ml. The low level of glucose and glu- tamic acid supported about 45% germination of_Mugg£ spores. and the high level of these compound supported about 80% germination. The relatively lower amount of germination occurring in the low concentration of nutrients may explain the occurrence of the inhibition zone. Moreover. the dynamic depletion of nutrients would continue after germination of 34 conidia. and the germ tubes would be exposed to a more and more depleted environment. Mycelia would not be expected to deve10p in such conditions. Observation of conidia of test fungi in inhibition zones supports this view; although some germination occurred. the germ tubes were unable to deve10p. To determine directly whether exhaustion of nutri- ents can cause inhibition zones on agar. the following ex- periment was devised. A special petri dish was prepared with the cover fitted with an inlet tube and the bottom with an outlet tube. The inlet tube was connected with sterilized plastic tubing to a separatory funnel contain- ing sterilized distilled water. The outlet tube was con- nected with sterilized plastic tubing to a sterilized flask. The entire system was autoclaved and maintained in a sterile condition. Twenty ml of 0.5% peptone-glucoes agar was poured into the special petri dish. A 10 mm wide strip of agar was removed from the plate. and replaced with a small amount of agar to seal the 2 semicircles of agar. The dish was tilted slightly and sterilized distilled water was allowed to drip into one end of the groove and run out the other end into the sterile flask. The rate of water flow 35 into the agar plate was about 0.5—0.8 m1 a minute and con- tinued for 7 days. At this time conidia of M. ramannianus or g. cingulata were sprayed on the agar surface. and water was again allowed to run through the system for 3 more days. At this time clear inhibition zones had developed adjacent to the groove (Fig. 7). The inhibition zones were about 10 mm wide at each side. The Spores of the test fungi in the inhibition zones had germinated poorly. When agar disks from these inhibition zones were transferred to fresh peptone agar. the conidia of both fungi germinated and developed mycelia. No contaminating microorganisms developed. Analysis of the agar in the zones showed that more than 90% of the glucose and amino acid were lost dur— ing the 10 day period of the experiment. Therefore. leach- ing of nutrients from agar can make inhibition zones which duplicate in every way those produced by actinomycetes. These results further indicate that the inhibition zones produced in agar by some actinomycetes. such as 3A and 8A. may be entirely due to the depletion of nutrients from the agar o 36 Fig. 7. Development of inhibition zones on leached agar. A special petri dish was designed to leach nutrients from peptone—glucose agar by allowing sterilized distilled water to run slowly through the groove between the two semicircles of agar. A clear inhibition zone developed following seeding with conidia of Glomerella cingulata. DISCUSS ION Inhibition of fungaL Spore germination in agar cul- tures by actinomycetes is generally thought to be caused by production of antibiotics. Inhibitory substances have been extracted many times from antagonistic cultures (2. 3. 4. 8. 25. 26). All antibiotics tested so far are diffusible in agar and should be detectable by diffusion assays (7. 15. 24). In this research. agar disks from inhibition zones of 9 actinomycete isolates or paper disks placed beneath in— hibition zones of these isolates. made new inhibition zones. when placed on fresh. seeded agar. thus demonstrating the presence of diffusible inhibitory substances. Eight of the same 9 isolates also produced diffusible antibiotics in liquid cultures. However. several findings indicated that 9 other actinomycete isolates that made inhibition zones in agar did not produce antibiotics. Agar disks from inhibi- tion zones when transferred to fresh seeded agar did not produce new inhibition zones. Negative results were also obtained with paper disks placed beneath the inhibition zones. 37 38 Many reports indicate that some cultures that showed antagonism on agar media failed to produce antibiotics in liquid media (7. 17. 21. 22. 24. 27). This research also showed that certain actinomycetes which produced inhibition zones against fungi failed to produce detectable antibiotics in agar or liquid cultures. There are some reasons for con- sidering the possibility that these actinomycete cultures may inhibit fungi by inducing a deficiency of nutrients in the agar. In this research. conidia of g. cingulata. which do not require exogenous nutrients. germinated when agar disks from the original inhibition zones were transferred to sterile petri dishes without agar. Conidia also germin- ated very well when incubated directly in 3-day old cultures of these actinomycetes grown in liquid media. Conidia of ‘M. ramannianus. which required ex0genous nutrients. germin— ated when nutrients were added to agar disks transferred from original inhibition zones. Actinomycetes are able to utilize a variety of com- plex organic compounds. Carboyhydrates and nitrogen sources. including glucose and amino acids. are known to rapidly utilized (2. 18. 23. 25. 27). Evidence was obtained that 39 glucose and glutamic acid were rapidly lost from agar media adjacent to actinomycete colonies. Results with leached agar showed that agar artifi— cially deprived of nutrients failed to support fungal growth. Almost all nutrients were leached from adjacent agar when water was allowed to run slowly between 2 semi- circles of agar. This area failed to support spore germin- ation and mycelia growth. The inhibition zone which re- sulted closely resembled those produced by actinomycetes. Therefore. inhibition zones produced by certain actinomy- cetes may be due entirely to depletion of nutrients from the agar. The fact that agar in the inhibition zones pro— duced by 2 antibiotic-producing actinomycetes was also rapidly deprived of nutrients raises a question as to the relative importance of antibiotics and nutrient depletion in production of inhibition zones by actinomycetes which produce antibiotics. Although 18 actinomycetes lysed fungal mycelia. in no case did agar disks from lytic zones make new lytic zones on fresh fungal cultures. However. new inhibition zones were made by those isolates shown to produce antibiotics. Therefore. the production of lysis zones can not be 4O attributed to antibiotics or to any demonstrable diffusible lytic factor. The role of nutrient deprivation in inducing autolysis of fungi in soil was shown by Lloyd and Lockwood (12). In view of the ability of 4 of the actinomycetes to effectively deplete agar of nutrients. possibly lytic zones on agar result from nutrient deprivation. These results also suggest the possibility that nu— trient competion as an antagonistic mechanism in agar may occur not only in actinomycetes. but also in bacteria and fungi. Much research in plant pathology has dealt with de- termination of numbers of antagonistic microorganisms in soils (6. 27). In work of this type. attempts are often made to correlate these populations with incidence of di- sease in certain soil types or following some kind of soil treatment. Assessment of number of antagonists and degree of antagonism are done in agar tests. It is usually stated or implied that these data provide a measure of the number of organisms capable of producing antibiotics active against a given pathogenic fungus. The results of this research suggest that interpretation of these kinds of data based on antibiotic production may be erroneous without further exam— ination of the mechanism involved. L ITE RATURE C I TED Bartholomew. W.. A. I. Rachlin. W. E. Scott. L. H. Sternbach and M. W. Goldberg. 1951. The isolation of three new crystalline antibiotics from Strepto- myces. J. Am. Chem. Soc. 73:5295-5298. Brian. P. W. 1951. Antibiotics produced by fungi. Bot. Rev. 17:357-430. Brian. P. W. 1957. The ecological significance of antibiotic production. ‘Ig 7th Symp. Soc. Gen. Microbiol. (Ed.. Williams. R. E. 0.. and C. C. Spicer). p. 168-188. Emerson. R. L.. A. J. Whiffen. N. Bohonos and C. Deboer. 1946. Studies on the production of antibiotics by actinomyces and molds. J. Bact. 52:357—366. Florey. H. W.. E. Chain. N. G. Heatley. M. A. Jennings. A. G. Sanders. E. P. Abraham and M. E. Florey. 1949. Antibiotics. Oxford University press. Vol. 1. 628 p. Kaufman. D. D.. and L. E. Williams. 1965. Influence of soil reaction and mineral fertilization on num- bers and types of fungi antagonistic to four soil- borne plant pathogens. PhytOpathology 55:570-574. Isheda. N.. T. Shiratori. S. Okamoto and I. Myazaki. 1951. The paper chromatography of antibiotic sub- stances from actinomyces. J. Antibiotics (Japan) 3:880. Johnson. L. F.. E. A. Curl and H. A. Fribourg. 1964. Methods for studying soil microflora-plant disease relationship. Burgess Publishing Co. 178 p. 41 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 42 K0. W. H.. and J. L. Lockwood. 1967. Soil fungis— tasis: relation to fungal Spore nutrition. Phyto- pathology 57:894-901. Lingappa. B. T.. and J. L. Lockwood. 1964. Activation of soil microflora by fungus spores in relation to soil fungistasis. J. Gen. Microbiol. 35:215-227. Lockwood. J. L. 1964. Soil fungistasis. Anu. Rev. PhytOpathology 2:341-362. Lloyd. A. B.. and J. L. Lockwood. 1966. Lysis of fungal hyphae in soil and its possible relations to autolysis. Phytopathology 56:595-602. Meredith. C. H. 1944. The antagonism of actinomyces to Fusarium oxysporum cubense. Phytopatholoqy 34: 426-429. Moore. 8.. and W. H. Stein. 1954. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211: 907-913. Murayama. O. 1959. Fundamental study on the disc method of antibiotic sensitivity test. J. Anti- biotics (Japan) 12:263-283. Nakhimovskaia. M. I. 1939. The antagonism between actinomycetes and soil bacteria. Microbiologia (U.S.S.R.) 6:131—157. Perlman. D. 1953. Physiological studies on the actin- omycetes. Bot. Rev. 19:46-97. Porter. C. L. 1924. Concerning the characters of certain fungi as exhibited by their growth in the presence of other fungi. Am. J. Bot. 11:168-188. Routien. J. B.. and A. C. Finlay. 1952. Problems in the search for microorganisms producing antibiotics. Bact. Rev. 16:51-67. 20. 21. 22. 23. 24. 25. 26. 27. 28. 43 Schatz. A.. and E. L. Hazen. 1948. The distribution of soil microorganism antagonistic to fungi patho- genic for man. Mycologia 40:461-477. Stessel. G. P.. C. Leben and G. W. Keitt. 1953. Screening tests designed to discover antibiotics suitable for plant disease control. Mycologia 45:325—334. Vanek. Z.. and Z. Hostalek. 1965. Biogenesis of antibiotic substances. Academic Press. 324 p. Waksman. S. A. 1945. Microbial antagonisms and anti- biotic substances. The Commonwealth Fund. 350 p. Waksman. S. A. and H. A. Lechavalier. 1962. The Ac- tinomycetes. Vol. III. Antibiotics of Actino- mycetes. Williams & Wilkins Co. 430 p. Waksman. S. A. 1963. The actinomycetes and their anti— biotics. Adv. Appl. Microbiol. 5:235-315. Waksman. S. A. 1967. The actinomycetes. a summary of current knowledge. Ronald Press. 280 p. Wood. R. K. S.. and M. Tveit. 1955. Control of plant diseases by use of antagonistic organisms. Bot. Rev. 21:441-492. Zoltan. B.. V. Betina and N. Pavel. 1964. Paper chromatographic classification of antibacterial and antifungal antibiotics produced by fungi isolated from soils in Indonesia. J. 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