W W m MM ‘J H “HH “3“ ‘11 A} ‘H HM" H ‘r ‘I ‘M w ‘H ENDUCTMN OF CHLAMYDOSPORE FESRMATEGN KN FUNGS 1N ASA? MEDIA BY STREPTGMYCETES H { H H i \H' \ ‘Fhesis fo: the {legvee of M. S: MECHEQW SLME UWERSEW MAMA 2W. 9mm E. 9 73 " (ax-r I"I LIBRARY :, '. Michigan State University _, mites '9; "DAB 3. sons" 800K swam mu. a LIBRARY Bl NDERS ABSTRACT INDUCTION OF CHLAMYDOSPORE FORMATION IN FUNGI IN AGAR MEDIA BY STREPTOMYCETES BY Marina Inn Chiang Each of thirty Streptomyces isolates from soil induced chlamydospore formation by Fusarium solani f. Sp. phaseoli isolates 20 and 151, E, solani f. sp. pisi and Mucor ramannianus in co-culture with the fungus. All iso- lates also inhibited fungal growth on the co-culture plates. Sixteen isolates produced substances in solid and liquid cultures which were capable of inducing chlamydospore.for- mation and inhibiting germination of growth of the fungus. Agar discs from the area adjacent to streptomycete colonies, when transferred to fresh medium, induced chlamydospore for- mation in all four fungi.- ChlamydOSpore-inducing substances from streptomycetes were detected when paper discs impreg- nated with sterile filtrates of liquid cultures were placed on.agar seeded with E. solani f. sp. phaseoli. The chlamydOSpore-inducing substances were extraced from liquid cultures of two selected isolates with amyl alcohol. The Marina Inn Chiang extract from one of the cultures inhibited macroconidial germination and induced chlamydOSpore formation from unger- minated macroconidia. The other inhibited germling growth followed by chlamydospore formation from the germlings. The remaining fourteen Streptomyces isolates failed to produce detectable chlamydospore—inducing substances in culture, and the possibility that these isolates induced chlamydospore production by nutrient deprivation was examined. When nutrients were rapidly exhausted from an agar medium by aqueous leaching, 100% chlamydospore formation was induced within 6 days. When four Streptomyces isolates, two of which produced chlamydospore-inducing substances, and two of which did not, were grown on agar containing only glucose and glutamic acid, 90% of both compounds were exhausted within the area of chlamydospore formation after six days. Therefore, nutrient deprivation in co-culture seemed to be the mechanism by which chlamydospore formation was induced for those Streptomyces isolates which do not produce chlamydOSpore-inducing substances. INDUCTION OF CHLAMYDOSPORE FORMATION IN FUNGI IN AGAR MEDIA BY STREPTOMYCETES BY Marina Inn Chiang 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 1973 to my parents ACKN OWLE DGMENT S 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. W. G. Fields and Dr. M. V. Wiese for their critical evaluation of the manu— script. I would also like to extend my appreciation to all of my laboratory-mates for their assistance. iii TABLE OF CONTENTS N-Page LIST OF TMLES O O O O O O O I O O O O O O O O O C O C v LIST OF FIGURES O O O O O O O O O O O O O O O O O O 0 Vi INT R0 DUCT ION O O O O O O O O O O O O O O O O O O O O I l LITE MTURE REVIEW 0 O O O O O O O O O O O O O O O O O 3 MTHODS AND MATERIALS 0 O O O O O I O O O I O O O O O 7 Preparation of Streptomycetes and Fungi . . . . . . 7 Detection of ChlamydOSpore-Inducing Substances . . . 8 Utilization of Nutrients in Agar by Streptomycetes O O O O O O O O O O O O O O O O O O 10 Induction of Chlamydospore Formation on LeaChed S and O O O O I O O O O O O O O O O O O O O 10 MSULTS O O O O O O O O O O O O O O O O O O O O O O O 12 The Characteristics of Chlamydospore Induction by Streptomycetes O O O I O O O O O O O I O I O O 12 Production of Chlamydospore-Inducing Substances in Solid Culture . . . . . . . . . . . . . . . . . 16 Production of Chlamydospore-Inducing Substances in Liquid Media . . . . . . . . . . . . . . . . . l9 Nutrient Status of Agar Adjacent to Streptomyces Colonies . . . . . . . . . . . . . . 24 Induction of Chlamydospores Formation under Leaching Conditions . . . . . . . . . . . . 25 DISCUSSION 0 O O O O O O O O O O O O O O C O O I O O O 2 7 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O O 31 iv Table LIST OF TABLES Page Chlamydospore-inducing and inhibitory characteristics of streptomycetes toward Fusarium solani f. sp. phaseoli isolate ISI and Mucor ramannianus in nonbuffered agar and qu uidfmedia . . . . . . . . . . . . 13-14 ChlamydOSpore-inducing and inhibition characteristics of selected strepto- mycetes towards Fusarium solani f. sp. phaseoli isolate—20 and F. son wi f. sp. p151 . . . . . . . . . . . . . . . . . . 15 Chlamydospore- inducing characteristics of streptomycetes toward Fusarium solani f. sp. phaseoli in buffered agar (pH 7. 0) . . . 17-18 Figure LIST OF FIGURES Page Rate of chlamydospore formation in macro- conidia of Fusarium solani f. sp. haseoli isolate 151 incfibated on Nuclepore membrane floated (a) on amyl alcohol extracts of cultures of three Streptpmyces isolates, (b) on distilled water, or (CT incubated on natural soil . . . . . . . . . . . . . . . . . 21 Loss of glucose from 3 mm diameter agar discs immediately adjacent to Streptomyces colonies growing on agar containing 0.2% glucose and 0.2% glutamic acid. Isolates 15 and 29 produced chlamydospore-inducing substances; isolates 10 and 11 did not. In the control, no streptomycetes were streaked on the agar . . . . . . . . . . . . . . . . . 21 Loss of glutamic acid from 3 mm diameter agar discs immediately adjacent to Stre - tom ces colonies growing in agar conta1n- ing 0.2% glutamic acid and 0.2% glucose. Isolates 15 and 29 produced chlamydospore- inducing substances; isolates 10 and 11 did not. In the control, no streptomycetes were streaked on the agar . . . . . . . . . . . . . 22 Chlamydospore formation by Fusarium solani f. sp. haseoli isolate 151, when agar discs conta1n1ng 0.2% glucose and 0.2% glutamic acid and seeded with macroconidia were placed in sand leached aseptically with distilled water, at the rate 1-1.5 ml/min. Control was macroconidia seeded on agar discs incubated on peptone-glucose agar . . . . . . . . . . . . . . . . . . . . . 22 vi INT RODUCT ION Resting structures of a number of soil-borne fungi have been reported to develop as a result of co—culture with other microorganisms, particularly bacteria. For example, production of sporangia by PhytOphthora cinnamomi (2, 30) as well as of chlamydospores of Fusarium solani were induced in co-cultures with soil bacteria in agar (9, 29) and in soil (9), whereas such structures were not pro- duced when the fungi were grown alone. Substances produced by the soil bacteria in these co—cultures were postulated as playing a morphogenetic role (9, 30). It is also possible that nutrient competition may have been involved in the induction of resting structures. Recently (22), it was reported that sudden withdrawal of exogenous nutrients from several form Species of E. solani in liquid culture caused rapid and abundant chlamydospore formation. Chlamydospore were also produced when the environment was deficient in energy but contained appropriate mineral salts (14). Previous work in our laboratory showed that some antagonistic streptomycetes inhibited the develOpment of fungi in agar media by production of antibiotics which could be detected in solid and liquid cultures, whereas others which failed to produce antibiotics caused inhibition by means of nutrient deprivation (13). Therefore, my research attempted to determine whether chlamydospore pro- duction in Fusarium spp. could be induced by co-culture with streptomycetes, and to elucidate the mechanism of such induction. LITERATURE REVIEW ChlamydOSpores provide the principal means of sur— vival of Fusarium spp. during unfavorable periods in soil. They are produced in response to a variety of stimuli, among which are staling product accumulation (25, 26, 27, 29), low C/N ratio of the culture medium (33), low concen- tration of a carbon source (5), specific chlamydOSpore- inducing substances (9, 31), starvation (12, 20, 21) or withdrawal of a carbon source (22), weak salt solutions in the absence of an energy source (14), and co-culture with other organisms (9, 30). Staling substances accumulated in the culture medium not only had a general inhibitive effect on E. gxyf sporum, but also had a positive morphogenetic effect with respect to chlamydospore formation (25, 27).. Following a period of rapid growth, normal mycelial elongation was inhibited and the hyphal apices abstricted conidia. As staling increased, swelling of the apices resulted in chlamydospore formation, and macroconidium germination was inhibited. Later, there was complete cessation of growth followed by mycelial autolysis. At this point, production of staling substances ceased and their concentration began to decrease the medium. It is not known whether the 3 separate morphological effects were all caused by a single substance or were due to several substances each with its own Specificity. Vankata Ram (30) found that certain bacteria stim- ulated chlamydoSpore formation by Fusarium solani on agar media. He suggested, without other evidence, that the phenomenon was due to the production of antibiotics by the bacteria. Three, and possibly four, substances obtained in aqueous extracts from one Salinas Valley soil induced chla- mydOSpore formation by one or more clones of Fusarium solani f. sp. phaseoli (8). Clones of the fungus responded differ- entially to each of the chlamydospore-inducing fractions. Extracts made from soil samples collected at different.times of the year varied in their capacity to induce-three clones to form chlamydOSporeS. Since non-sterilized extracts of soil induced the fungus to produce more chalmydOSpores than did sterilized extracts, soil bacteria were thought to be involved in the production of chlamydospores. In further work (9), isolates of three genera of soil bacteria (P527 taminobacter, Arthrobacter and Bacillus) induced chlamydo- spore formation in co-culture with the fungus. Substances isolated from culture filtrates of some of the bacteria showed Specificity in inducing chlamydOSpore formation by different clones of the E. solani f. Sp. phaseoli under axenic conditions.~ Therefore, Specific bacteria were thought to be responsible for producing chlamydospore- inducing substances in soil. Ford, gt_31. (10) also showed that a limited amount of energy source allowed chlamydospore production, whereas excess nutrients decreased the effect of chlamydospore— inducing substance. They interpreted their work as indicat- ing that the potential for chlamydOSpore formation was regulated primarily by the concentration of the chlamydospore- inducing substances and to a lesser extent by the amount of energy source. Other evidence indicates that carbon starvation may be a primary cause of chlamydospore formation. Low levels of carbon in the culture medium favored chlamydospore forma- tion by E. oxysporum f. Sp. cubense (33). Carlile reported that a low C/N ratio favored chlamydOSpore formation in F. oxysporum (5), but it was not determined whether the deter- mining factor was the prOportion of carbon to nitrogen or the actual carbon and nitrogen levels. Lockwood (21) pro- posed that as exogenous nutrients become exhausted, hyphal lysis and resting spore formation occur. For several Species of Fusarium, the sudden withdrawal of exogenous carbon source from a liquid culture medium (22), or incubation in the presence of suitable salts without a carbon source (14) favored chlamydospore formation. According to Lockwood microbial activity at the immediate surface of spores or other resting structures will keep the fungal prOpagales in a more or less continual state of nutrient deprivation (20). Deprivation of energy substrate per se does not explain all aspects of chlamydospore formation in E, solani f. Sp. phaseoli. For example, it doesn't explain why ster— ilized extracts of soil support greater chlamydospore for- mation in E. solani than sterilized water alone (8). In a recent study (24), chlamydospores of §.'Solani were formed in the same amount when washed nongerminated macroconidia incubated on Nuclepore filters were floated on dilute Na2804 solutions as on soil. Incubation in water or in phosphate buffer at pH 7.0 did not result in chlamydospore formation. Germlings of five Fusarium form species borne on membrane filters on NaZSO4 solution or water also formed chlamydo- spore rapidly. Chlamydospores also were formed from germ- lings incubated on acid-washed sterilized sand continually leached with water or phosphate buffer (pH 6.9), but fewer chlamydospores were formed without leaching. Therefore, chlamydOSpore production may require an environment deficient in energy but containing apprOpriate mineral salts. METHODS AND MATERIALS Preparation of Stre tomycetes and Fungi Thirty unidentified Streptomyces isolates were iso- lated from Conover loam soil from the Michigan State Univer- sity Farm (17) by means of soil dilution plates, using chitin agar (19) as a selective medium. The isolated selected differed in morphological characteristics and - colony color but none were identified to species. The iso- lates were maintained on yeast extract-maltose-glucose agar Slants (per liter: yeast—extract 4g, maltose 10g, dextrose 4g, agar 209). The fungi used were Fusarium solani (Mart.) Appel & Wr. f. sp. phaseoli (Burk.) Snyd. & Hans. isolates 20 and 151, Mucor ramannianus Mfiller and Fusarium solani f. sp. pigi (F. R. Jones) Snyd. & Hans. Tests for chlamydospore induction were made in petri dishes on peptone-glucose agar (per liter: peptone 5g, glu— cose Sg, agar 209) with and without 0.1 M phosphate buffer (pH 7.0). Streptomyces isolates were streaked on the medium and incubated at 24C for 7 days. Then spore suspen- sions of the test fungi were sprayed onto the agar surface with a medicinal atomizer attached to a compressed air line mounted on a ring stand with the nozzle 25 cm from the sur— face of the agar.' Approximately 2.5x105 spored were sprayed onto each plate. After 1, 2 and 3 days the plates were examined for chlamydOSpore formation. Agar discs adjacent to the Streptomyces colonies were removed aseptically with a 3 mm diameter cork borer, and stained with phenolic rose bengal and observed with a microsc0pe. Chlamydospore pro— duction was expressed as the percentage of macroconidia or germlings forming one or more chlamydospores. Detection of Chlamydospore- InducingSubstances To detect chlamydospore-inducing substances found in agar media, agar discs 5 mm in diameter were cut with a sterile cork borer from glucose-peptone agar adjacent to Streptomyces colonies and transferred to fresh peptone agar (per liter: peptone 59, agar 209) plates. After 24 hours conidial suspensions of the four test fungi were sprayed 'onto the agar surface. One, 2, 3, 4, 5 and 6 days later the induction of chlamydospore formation was observed in the manner previously described. For the production of chlamydospore-inducing sub- stances in liquid media, streptomycetes were cultured in dup— licate, shaken 125 ml Erlenmyer flasks containing 25 ml glucose-peptone broth (per liter: peptone 59, glucose 59) for 7 days at 24C. Filter paper antibiotic assay discs (13 mm diameter) were impregnated with sterile filtrates by applying 0.1 ml at a time, then alternately air-drying the discs. The discs were then placed on the surface of peptone agar plates and allowed to stand for 12 hours, when conidial suSpensions of the four test fungi were sprayed onto the plates. The induction of chlamydospores was observed micro- scopically 1, 2, 3, 4, 5 and 6 days after application of the fungal spores. Uninoculated liquid medium treated in the same way as the culture served as the control. Liquid cultures of three selected Streptomyces iso-V lates were passed through Millipore filters (0.22u) and the filtrates were extracted with an equal volume of amyl alco— hol. The amyl alcohol extracts were first dried, then redissolved in 5 ml sterilized distilled water. The extracts were placed into small glass petri-dishes, and Nuclepore- membrane filters (10 mm diam., 0.5 0 pore size) bearing washed macroconidia of F. solani f. sp. phaseoli isolate 151 were floated on the distilled water or placed on Conver loam soil. After incubation for 0.25, 0.5, l, 3, 4, 5, 6 and 7 days, one filter was removed from each of the three substrates. To observe chlamydospore formation, the fungi were stained with phenolic rose bengal, and the membranes made transparent by mounting them in clove oil on glass slides (14). 10 Utilization of Nutrients in Agar by Streptomycetes An agar medium was prepared in 0.1 M phosphate buffer (pH 7.0) and contained, per liter: glucose 29, glu- tamic acid 29, agar 209 or 309. Two layers of agar were prepared: a bottom layer (5 m1 of 309 agar/liter) was poured in the petri-dishes. After hardening, the t0p layer (10 ml of 209/liter) was poured onto the bottom layer. Streptomyces isolates 10, ll, 15 and 29 were streaked on the agar surface. This medium supported good growth of F. solani f. Sp. phaseoli isolate 151 and the streptomycetes. On the lst, 2nd, 4th, 6th and 8th day, 6 agar discs 3 mm in diameter were removed from the tOp layer of agar adjacent to the Streptomyces colonies and melted in 3 ml distilled water. Glucose in the agar was measured colorimetrically at 420 mu. Glucose at concentrations of 5, 10, 15, 20 ug/ml was used as a standard. Glutamic acid was assayed colorimetrically at 570 mu using the ninhydrin reagent (23). Glutamic acid at concentrations of 2, 3 and 4 ug/ml was used as a standard. Induction of Chlamydospore Formation on Leached Sand Discs 3 mm in diameter were cut from glucose-glutamic acid agar (per liter: glucose 29, glutamic acid 29, agar 209) and inoculated with macroconidia of F, solani f. sp. phaseoli isolate 151. The seeded discs were immediately transferred 11 to the surface of a sand bed in a leaching apparatus (15). This was composed of a separatory funnel equipped with a sealed-in dripping tip to maintain a constant flow rate. The funnel stem was connected by plastic tubing to a needle valve, then to a glass petri-dish fitted with an inlet in the lid and an outlet at the bottom on the Opposite side. The dish contained a 5 mm layer of acid—washed silica sand. The separatory funnel was filled with distilled water. The entire apparatus was sterilized by autoclaving before use. Control discs were incubated on glucose-peptone agar. After leaching l, 2, 3, 4, 5 and 6 days, two discs were removed from the sand bed, stained with phenolic rose bengal, and observed microsc0pically. ChlamydOSpore production was determined in 100 macroconidia on each disc. RESULTS .Ebe Characteristics of. Chlamydospbre”Indu¢tion By Streptomycetes All of the thirty streptomycete isolates induced chlamydospore formation when co-cultured with F, solani f. Sp. phaseoliisolates 20 and 151, E, solani f. Sp. pi§i_and M, ramannianus (Tables 1, 2). Twenty-three of the isolates also inhibited macroconidial germination resulting in clear zones surrounding the streptomycete colonies. Chlamydo- spores were formed within-3-5 days from mycelia around the edges of the inhibition zones as well as from ungerminated macroconidia near the periphery of the inhibition zones. The other seven streptomycete isolates did not inhibit macroconidial germination but did inhibit germling growth, producing a partially clear zone. Chlamydospores were formed throughout the area of growth inhibition as well as at the edge of the zone. Apparently mature chlamydospores were observed as early as one day after macroconidia were applied to the agar.- Since the pH of the non-buffered agar medium around the Streptomyces colonies increased from 6.9 to 7.6-8.3, Chlamydospore induction of E. solani f. sp. phaseoli 12 13 I + + ea I + + ma I + + NH I + + Ha I + + OH I + + m I .+ + m H I + + n - + I .. I + + m I + + v I + + m I + + N I + + H I I MI Honucou mmusuaso pwswfid I ....cofluunodd...GOHuflnflscfi mDMHOmH maouo can Hmmm SR mnouomm whommOUhstcu cowumcwsumw mcflosocflImHommOUNEmHsu chauHSOIoo noon: no musuaso mmomfioummuum Edwummsm+mcuwsoummuum .ma we pfldvfla cam Hmmm wmnmmmsncoc SH mSSMHSSMEmH Mons: can and mumHomH HHOmmma ..mm .m Hanaom.finfiHMmsm_UH930# . . . moumohfioummnum mo mowumwumuomumno unouflnwscw can mcHUSpcflIwHommO©mEmHno .H magma l4 .nuzoum was» Sums mo cofluflnflan .SOSHUSUSH cucumOpmamano Ho Q cofluflnfinnfl on u I “aoHuosccw muommOUNEmano Ho aofluwnflncfl cowumcflfiumo u +m + + 3+ om + + n+ mm + + 3+ mm + + 3+ 5N HHH + + 3+ mm + + Q+ mm + + 3+ vm + + 7+ mm + + .+ mm + + + Hm + + + on + + + ma HH + + + ma + + + NH + + + ma + + + ma 15 .nugoum was» Sumo mo aofluflnaaaH n .coHHOSpcfl chommocwfimano no coauflnwscfl o: u I “coauospcfi chommOpmfimano Ho coflufinflncw coflumnflanow n +m + + Q... mN HHH + + + ma HH I + + OH H I + + m I I MI Monacoo monsuauo pwswfla . .. ...... .doauuancfl..4d0Hanflznw mumaomH macho can Hmmm SH muouomu cnommochfimHnU SOHDMGHEHow mSAOSpcflIcuommOmemaco musuflmOIoo ousuaso chSEOHQWWHm msmcsm+mmomaoumwuum .mmwm .mm .m “SMHOm am can om mumaomfi flaocmmnm .mm .m ficmaom ESHHmmnm mpnmzou mmuwomaoumwuum pmuomacm no moaumfinmuomumno whoufinwncw can mcHOSoGHImHommO©MEMH£U .m canoe 16 isolate 151 was retested using buffered agar (pH 7.0) to determine if chlamyd03pores were pH-induced. Chlamydospores were formed on the buffered plates at the same rate as on non-buffered plates (Table 3); on the buffered agar, the pH remained at 7.0. The size of inhibition zones produced by the same isolates was Similar in both buffered and non- buffered agar with the exception of isolates 10, 11 and 23 which produced smaller zones in buffered agar. The aberrant result with these three isolates appeared to be due to poor growth of the streptomycetes in the presence of phOSphate rather than to any effect of pH. In another experiment Fa‘solani f. sp. phaseoli iso- lates 20 and 151, F, solani f. Sp. pigi and M, ramannianus failed to form chlamydOSpores in agar buffered at pH 7.0, 7.3, 7.7 and 8.3 with 0.1 M phosPhate buffer, even though growth at all pHs was substantial. Therefore, pH of the agar seemed to have no effect on chlamydOSpore formation. Prgduction of Chlamydospore- Inducin CSubstances in SoIig Culture To determine whether the Streptomyces isolates pro- duced substances which induced formation of chlamydospores in the agar medium, discs cut from the area adjacent to the Streptomyces isolates were transferred to fresh peptone agar plates seeded with conidia of F. solani f. Sp. Bhaseoli isolate 151 or Mucor ramannianus. Agar discs from the l7 +- + MV' 0‘ I" -+ + -+ + -+ + -+ + -+ + -+ + -+ + 12.0 + -+ + -+ + -+ + -+ + -+ + HNMVLOKDFCDGOH + N r-II-lr-lr-II-l Gem I Ml Honucou GOADUSpSH cowuflnfiscw mDMHOmH msono ouommovwfimano SOHDMGHEHow Acmummmsncsv . _ Homo wunuHSOIoo .. Aummm pwummmnflv +moommoumwuum mo mm musuHSOIoo ESHHMmsm+mmoMEoummHum ..»~0»N.mmv.wmmw.udku%%inpdw.wwccmn£.Hnmw.rw.HSMHom ESAHMmsm pnm30u mmumomfioummuum mo mowumflnouomumso mSAOSOSHImHommocmEMHSU .m magma .suzonm masu Sumo mo GodufinfiscHo .Esflpofi pmummanIcos so #9:“ cmnu mcoN Gofluflnflscfl HmHHmEmn .coHDOSUcfi chomm0paamaao Ho 18 :oHufinflncw o: u I «cofluospcfl whommochewano Ho cofluwnflncfl coflumcflfinmw u +0 m.h + 0+ om m.h + 0+ mm o.m + 0+ mm N.m + 0+ 5N o.m + 0+ wN o.m + 0+ mm m.m + 0+ VN m.b ................ + ...... Q+ . mm m.h + + NN m.h + + HN m.b + + ON o.m + + ma N.@ + + ma o.m + + 5H m.m + + mH m.h + + mH m l‘ + + 19 medium adjacent to isolates 15, l6, l7, l8, 19, 20, 21, 22 and 23 caused new inhibition zones surrounding them, and chlamydOSporeS were formed in the mycelium and germ tubes at the edges of such zones (Table 1). Discs from near iso- lates l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13 and 14 neither caused chlamydOSpore formation nor inhibited germina- tion or growth. Agar removed from the vicinity of a third group of isolates, 24, 25, 26, 27, 28, 29 and 30, inhibited germ tube growth which was followed by chlamydospore forma- tion both from germ tubes and conidia. Production of Chlamydospore- InducingSuBstances in Liquid Media To further test whether chlamydospore-inducing sub- stances were produced by the Streptomyces isolates, all 30 isolates were grown in a liquid medium. Sterile culture filtrates of isolates 1-14, which failed to produce chlamydo- Spore-inducing substances on agar media, also failed to induce chlamydospore formation in F. solani f. Sp. phaseoli isolate 151 and M, ramannianus (Table l). The selected isolates 10 and 11 also had no effect on g, solani f. Sp. phaseoli isolate 20 and F, solani f. Sp. pigi. The other 16 isolates, which produced chlamydospore-inducing substances on agar media, also induced chlamydospore formation of F, solani f. Sp. phaseoli isolate 151 and M, rammanianus. Fil- trates from 9 of these isolates (15-23) also inhibited 20 macroconidial germination surrounding the paper discs, whereas filtrates of 7 other isolates (24-30) had no effect on ger- mination but inhibited germling growth (Table 2). Culture filtrates from isolates 15 and 29 also caused the same effects on Fusarium solani f. Sp. phaseoli isolate 20 and E, solani f. Sp. pi§i_(Table 2). Inhibition zones for M, ramannianus usually were larger than those of other fungi. Culture filtrates of three selected Streptomyces isolates were extracted with amyl alcohol. One (isolate 15) produced chlamydospore-inducing substances and completely inhibited macroconidial germination, another (isolate 29) produced chlamydospore-inducing substances but inhibited only germ tube growth, and the third (isolate 11) did not produce chlamydospore-inducing substances. The extracts were tested for induction of chlamydospore formation by E. solani f. Sp. phaseoli isolate 151 borne on Nuclepore fil- ters. The extract from isolate 15 induced chlamydospore formation within 12 hours, and 100% of the macroconidia had formed chlamydospores within 48 hours (Figure l). Chlamydo- Spore formation in the extract of isolate 29 was first ob- served in 6 hours, and 100% of the macroconidia had formed them within 24 hours. Chlamydospore formation in the extract from isolate 11 was first observed in 48 hours, and at the 7th day there was 70% chlamydospore formation. 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