EFFECT OF PHYSICO-CHEMWICAL ENVIRONMENT ON SPHERULATION OFCOCCIDIOIDES IMM1ITIS IN A CHEMICALLY DEFINED MEDIUM

JOHN LAY CONVERSEFort Detrick, Frederick, Maryland

Received for publication May 16, 1956

Coccidioides immitis undergoes a completelydifferent type of growth in vivo from that invitro. The parasitic form, or spherule, appearsin animal tissue as a non-budding, sphericalstructure 20 to 80 ,u in diameter with a thick,refractile, double wall, and is filled witlh numer-ous small endospores 2 to 5 ,u in diameter. Thesaprophytic form appears in laboratory mediaas mycelium with branching, septate hvphaewhich fracture into numerous thick-walled,rectangular, spherical, or ellipsoidal arthrospores2 to 4 ,u in diameter.

Observation of the parasitic form of C. i7mnitisin solid or semi-solid complex media has beenreported by several authors. AMacNeal and Tay-lor (1914) observed multiplication of spherulesin infected pus when added to ascitic fluid orgelatinized horse serum containing sterile kid-ney slices. Lack (1938) noted development ofspherules from arthrospores inoculated intoHall tubes containing glucose broth and par-tially coagulated egg albumin. Baker and MIrak(1941) observed the development of spherulesin a variety of solid media when active growthhad ceased as a result of drying of the media.Burke (1951) reported the appearance of spher-ules in a partially defined solid medium contain-ing coconut milk. Conant and Vogle (1954)observed spherules in cultures transferred toSaboraud's medium following exposure to"Tween 80." Lubarsky and Plunkett (1955)were able to produce spherules in Tyrode'ssolution, supplemented with chick-embryo ex-tract and serum and aerated periodically withmeasured amounts of oxygen and carbon dioxide.This laboratory was the first to report produc-tion of spherules in a chemically defined liquidmedium (Converse, 1955).The work reported below represents further

studies on the formation of spherules in a chemi-cally defined liquid medium (figures 1 to 6).The following factors were investigated: thetotal concentration of nutrients in the medium,

the hydrogen ion concentration of the medium,the temperature, aeration, and exposure tolight during incubation, and the preparation ofinoculum.

MATERIALS AND METHODS

Medium. The experimental medium, unlessotherwise indicated, was composed of 0.022 Mglucose, 0.016 M ammonium acetate, 0.003 Meach of mono-potassium and di-potassium phos-phate, 0.0016 Ai magnesium sulfate, 1.24 X 10-5Ai zinc sulfate, 2.4 X 10-4 M sodium chloiide,2 X 10-5 M calcium chloride, 1.4 X 10-4 Msodium bicarbonate, and distilled water, with afinal pH of 6.6. This medium is referred to here-after as basal spherule medium. The mediumwas dispensed in 50-ml volumes into 250-mlpyrex glass Erlenmeyer flasks, which were closedwith i ubber stoppers, each bearing a thistletube packed with gauze-covered cotton, andwas sterilized by autoclaving for 15 min at121.5 C.Inoculum. C. itmimitis strain Ml1, a rodent

isolate from Airizona, was grown for 28 days at 34C with shaking, in a liquid synthetic medium(Basal # 2, Roessler et al., 1946 + 4 ppm Zn+as ZnSO4) and stored at 5 C until use. Eachflask of experimental medium received 0.1 mlof the inoculum or approximately 1 X 107viable particles (short chains of arthrosporesand hyphal fragments).

Incubation. Experimental cultures were in-cubated for 72 hr at 34 C on a reciprocal shakingmachine operating through a 4t,-in stroke atapproximately 96 excursions per min.Assay procedures. Spherule yields were deter-

mined by microscopic examination of lacto-phenol-blue wet mounts made from culturematerial. Comparative rather than absolutemeasurements were used, due to the lack of asatisfactory method for separation of spherulesfrom other growth structures. The rielativeamount of spherules to total growth was esti-

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Figures 1 to 6 show development of Coccidioides immitis spherules from arthrospores and release ofendospores into substrate.

Figure 1. Arthrospore inoculum. X 300. Figure 2. Young spherule showing start to cleavage. X 300.Figure 3. Spherules in later stage of development showing an increase in cleavage planes. X 435Figure 4. Spherules with maturing endospores in evidence. X 300. Figure 5. Spherules with fracturedcell-walls. Endospores are being released into substrate. X 435. Figure 6. Appearance of culture fol-lowing endospore release. X 167.

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mated for each culture and assigned a numberon an arbitrary scale of 0 to 8, with increasingnumbers indicating increased spherule formation.With this method, comparative measurementscould be made among cultures.

Total growth (hyphae, arthrospores, spherules,and endospores) of cultures was determinedeither by macroscopic appearance of cultures,using comparative estimations on a scale of 0to 8, or by viable plate counts. In the lattermethod, the pour-plate technique was used witha plating medium composed of peptone, 1 percent; glucose, 2 per cent; yeast autolysate, 0.1per cent; and agar, 2 per cent; with final pH of6.9 to 7.2. Plates were incubated for 48 hr at34 C.

Light intensities were measured in foot candleswith a General Electric exposure meter (PH77 C, Type DW-48).Hydrogen ion concentrations were deter-

mined with the glass electrode.

RESULTS

Dilution of medium. Previous observations(Converse, 1955) indicated that the total con-

centration of nutrients in the chemically, de-fined medium influenced the in vitro growth ofthe parasitic phase of C. immitis. To determinethe optimum concentration, duplicate flasks of a

liquid synthetic medium (Basal #2, Roessleret al., 1946, plus 4 ppm of Zn+ as ZnSO4) having3.36 per cent total solids were diluted with dis-tilled water to 90, 80, 60, 20, 15, and 10 percent of the specified total solids content andinoculated with C. immitis strain M1.

TABLE 1

Effect of dilution of medium on growthand spherulation

Per Cent Solids inMedium (Total Viable Fragments* Spherule Yield6

Nutrient Content)

X 106 per ml

3.36 113 03.03 74 02.69 72 Ot2.02 49 Ot0.67 16 20.48 14 40.34 6 5

* Reported as averages of duplicate flasks.t Occasional spherules noted (very scarce).

TABLE 2Effect of pH of medium on growth and spherulation

pH Visual Estima- Sheruletion of Total yildInitial Terminal Growth* Seld

3.0 3.35 0 04.0 4.27 0 05.0 6.23 0-it 06.0 7.20 4 26.6 7.40 4 28.0 7.63 4 29.0 7.70 4 1-210.0 7.93 3-4 1

* Reported as averages of 2 to 4 flasks.t No sporulation of any type in these cultures.

As shown in table 1, spherule formation in-creased progressively with dilution of the me-dium, beginning at the 80 per cent level andbecoming very apparent at the 20 per cent level.However, a proportionate decrease in growthof the cultures also resulted. Consideration ofthese opposing effects on spherule yield led tothe selection of a medium with 20 per cent of thespecified total solids (a five-fold dilution of theoriginal medium) as a basal spherule mediumfor further experimentation. Repeated observa-tions indicating the stimulatory effects of NaCl,CaCl2, and NaHCO3 on spherule developmentled to their inclusion in the basal spherule me-dium at the concentrations reported in the sec-tion on materials and methods.Hydrogen ion concentrations of the medium.

Previous observations in this laboratory indi-cated that optimum growth of the saprophyticor mycelial phase of C. immitis in a glucose-ammonium acetate-inorganic salts mediumwas obtained if the initial pH was adjusted tobetween 7.0 and 7.5. It was also noted that thefinal pH of the cultures was always approxi-mately 7.7 regardless of the initial pH of themedium. The optimum pH range for growth ofthe parasitic phase was established by inocu-lation of duplicate flasks of basal spherule me-dium adjusted to pH values of 3.0, 4.0, 5.0, 6.0,6.6, 8.0, 9.0, and 10.0 before sterilization.The effect of initial pH of the medium on

growth and spherule production is summarizedin table 2. No growth occurred below pH 5.0 andno sporulation of any type (arthrospores, chlamy-dospores, or endospores) occurred below pH

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6.0. Maximum spherule yields were obtainedat 6.0, 6.6, and 8.0, with no significant differenceamong the three levels. A few spherules werenoted in media with initial pH values as highas 10.0. Total growth of the cultures was notsignificantly different at any pH level between6.0 and 10.0.

Temperature of incubation. The importance ofincubation temperature in the in vitro growthof the parasitic phase of other dimorphic fungi,Histoplasma caps-ulatum and Blastomyces der-matitidis, has been noted by Salvin (1949a, b),who was able to grow the yeast phase of bothorganisms at 37 C, but not at 25 C. It was thoughtthat an incubation temperature close to that innormal animals might also stimulate in vitrospherule formation of C. immitis. To determinethe effect of incubation temperature on spherula-tion of this organism, duplicate flasks of basalspherule medium were inoculated and incubatedwith shaking at 25, 26, 30, 34, 37, and 41 C.

Microscopic examination of the cultures after72 hr of incubation indicated (table 3) that anincrease in temperature from 26 and 37 C wasparalleled by an increase in spherule formation.No spherules were formed at 25 or 26 C. Approxi-mately equal growth was obtained at all temper-atures except at 41 C, which inhibited germ-ination of the inoculum. No spherules wereevident in the 41 C cultures at 72 hr, butcontinued incubation at this temperature for 10to 14 days resulted in almost 100 per cent con-version of the arthrospore inoculum to spher-ules, without intermediate mycelial growth.

Aeration of cultures. Observation of Mader(1943), Henry and Anderson (1948), Barnett andLilly (1950), and others, indicated that oxygentension, carbon dioxide content, and accumula-tion of volatile metabolic products in the medium

TABLE 3Effect of temperature of incubation on spherulation

Temperature of Incubation Spherule Yield*

C

25 026 030 1-234 337 641 0

* Reported as averages of duplicate flasks.

were important factors in the spoirulation ofvarious fungi. Conant (1941) observed the yeast-like or parasitic phase of H. capsulatum in sealedtubes of blood agar, but not in unsealed tubes.Bullen (1949) was able to grow the yeast-likeor parasitic phase of the dimorphic, pathogenicfungi, H. capsulatum, B. dermatitidis, and Sporo-trichum schenckii on peptone agar by controllingthe carbon dioxide content of the atmosphereabove the cultures. Since all of these factors maybe influenced by changes in aeration of culturesduring incubation, an attempt was made toestablish the effect of aeration on developmentof the parasitic phase of C. immitis in syntheticmedium.

Preliminary experiments, in which aeration ofthe medium was varied by changing either thediameter of the flask opening or the surface-to-volume ratio of the medium, resulted in a relativeincrease of spherule formation with decreasedaeration. Stationary incubation at 34 or 37 Cresulted in improved spherule production overthat of incubation with shaking at either of thesetemperatures. Stationary cultures containedtwice the number of spherules found in shakencultures, but the total growth was reduced by50 per cent. Also the length of incubation formaximum spherule production was 9 to 12 daysin stationary cultures, as compared with 3 to 5days in shaken cultures. Spherules in stationarycultures occurred in extremely long chains(figures 7 to 9), in contrast to the short chainsand single spherules noted in shaken cultures(figures 10 to 12).Attempts were made to increase spherule

formation and at the same time maintain normalgrowth levels by initiating growth with stationaryincubation and completing the growth cycle onthe shaker or by reversing this procedure. Twosets of replicate cultures were incubated at 34 C,one set in a stationary position and the other onthe shaker. At 24-hr intervals throughout a4-day period duplicate flasks were removed fromthe shaker and placed in a stationary position.At 48-hr intervals over a 12-day period duplicateflasks were removed from the stationary positionand placed on the shaker. The latter time in-tervals were longer since growth of stationarycultures was much slower. All flasks were ex-amined microscopically at 24-hr intervals betweenthe third and fourteenth day of incubation.Optimum spherule development was obtained in

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cultures completing a stationary period of in-cubation of 7 to 9 days during the initial stage ofgrowth. Cultures remaining on the shaker formore than 3 days, initially, were unsatisfactory,due to the gradual disappearance of spherulesafter the 3rd or 4th day. However, it was neces-sary to incubate cultures on the shaker for thefirst 2 or 3 days in order to maintain normalgrowth levels.

Exposure to liht. The effect of light on sporula-tion of various fungi often has been reported inthe literature. A stimulatory effect was noted byChristenberry (1938), Yarwood (1941), Timnicket al. (1951), and others. However, it was sus-pected that the opposite may obtain with theparasitic phase of C. immitis, since absence oflight during incubation would duplicate morenearly the normal conditions of spherule de-velopment in animal tissue. The effects of simu-lated daylight, twilight and darkness on spheruleformation and the time at which these conditionsexerted their greatest effect were determined inthe following manner.For the daylight condition, a reflector con-

taining two 15-watt daylight fluorescent bulbswas suspended 12 in above a shaker tray, to givea light intensity of 63 foot candles at the base ofthe flasks. Artificial twilight was maintained bypositioning a second tray on the shaker so thatthe reflected light from the fluorescent bulbsresulted in a light intensity of 4.5 foot candles.Complete darkness was maintained by placing aloosely fitting metal cover over a third tray,which excluded all light but did not interferewith aeration of the cultures.

Duplicate flasks of basal spherule mediumwere inoculated with strain Mll and incubatedat 34 C for 72 hr under conditions of continuouslight, darkness, and twilight. Other flasks werealternated between light and darkness and vice

TABLE 4Effect of light on spherulation

Light Exposure in Foot Candles during 72-hrIncubation Period Spherule

Yieldt0-24 hr 24-48 hr 48-72 hr

63 63 63 0-it63 63 0 1-263 0 63 1-263 0 0 1-24.5 4.5 4.5 30 63 63 3-40 63 0 40 0 63 4-50 0 0 4-5

* Reported as averages of 4-10 flasks.t Occasional spherules noted (very scarce).

versa at the end of each 24-hr interval of the72-hr incubation period. Data from microscopicexamination of the cultures are recorded intable 4. Complete absence of light was mostfavorable for spherule formation. Under condi-tions of intermittent illumination, spheruleformation was increased when darkness occurredduring the initial part of the incubation period.The rate of growth, or total growth of the culturesas estimated macroscopically, was not affected byvaried illmuination.

Preparation of inoctdum. The occasional failureof C. immitis to produce spherulesunder optimumconditions led to an investigation of the inoculumas a possible cause. Replicate flasks of basalspherule medium were inoculated with culturesof strain Mll grown for 28 days and stored at5 C for either 1 month or 4 months. Other flaskof the medium were inoculated with fresh culturesgrown for 4, 7, 14, or 28 days. All flask wereincubated with shaking at 34 C for 72 hr andexamined microscopically for spherule production.

Figures 7 to 9, and 10 to 12, show the appearance of spherules in shaken cultures, and stationarycultures, respectively.

Figure 7. Representative field of shaken culture. X 167.Figure 8. Enlarged view of culture in figure 7 showing detailed morphology of spherules in all stages

of development. X 300.Figure 9. Mature spherules. Note shrunken empty spherule wall in center following endospore re-

lease. X 435.Figures 10 and 11. Representative fields showing extreme length of spherule chains in stationary

cultures. X 167.Figure 12. Enlarged view of culture in figures 10 and 11 showing detailed morphology of spherule

chain. Note beginning of endospore release in two of the spherules. X 435.

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TABLE 5Effect of age of inoculum on spherulation

Age of InoculumSp;herule Yield

Growth at 34 C Storage at 5 C

days mo28 4 Ot28 1 228 0 214 0 47 0 54t 0 0

* Reported as averages of 2 to 4 flasks.t An-occasional spherule seen in these cultures.t No arthrospores present in this inoculum.

As indicated in table 5, extended storage at5 C was detrimental to the development ofspherules. Inocula grown for 7 and 14 days weresuperior to those grown for either 4 or 28 days.A complete lack of arthrospores in the 4-dayinoculum was noted on microscopic examinationof the various inocula, which suggested that,although younger inocula were more effective inspherule formation, the presence of arthrosporesin the inoculum was necessary.

DISCUSSION

The mechanism responsible for the develop-ment of either the parasitic or the saprophyticphase of C. immitis under different environ-mental conditions is not fully understood, norhave the physiological conditions necessary fordevelopment of the parasitic phase in vitro beenfully established. However, the data presentedabove provide evidence that spherulation in achemically defined medium must take place at acritical period early in the development of thecultures, beyond which growth of the parasiticphase will be unsuccessful. The rate of growth ofcultures may be of prime importance in establish-ing this critical point. This may explain thefailure of inocula devoid of arthrospores toproduce spherules (the extra time required forhyphal conversion to arthrospores and thence tospherules).

Aeration had a marked effect on rate of growth.Stationary incubation resulted in slower de-velopment of the cultures and approximately50 per cent of the total growth compared toincubation with shaking, but the relative spherule

yield was doubled. It was necessary to harvestspherules from shaken cultures not later than thefourth or fifth day of incubation, as a gradualdisappearance of the parasitic form obtainedthereafter, unless development was halted bystorage at 5 C. Conversely, cultures could beincubated in a stationary position for 14 days orlonger without ill effects. Disappearance of theparasitic form in shaken cultures was probablydue to a selective change in environment, such asexhaustion of specific nutrients or accumulationof metabolic products, which favored mycelialdevelopment and inhibited spherule development.The occurrence of these effects would be a func-tion of the rate of growth.The amount of oxygen or carbon dioxide

available to the organism during growth wasprobably an important factor in spherule de-velopment and could be directly affected byeither rate of growth or aeration. This is sup-ported by observations by Conant (1941) andBullen (1949) on the growth of the so-calledyeast phase of other dimorphic pathogenic fungiunder controlled carbon dioxide atmosphericconditions. Bullen was able to separate the effectsof oxygen from those of carbon dioxide and showthe oxygen tension was unimportant, but in-creased carbon dioxide content favored thegrowth of the parasitic or yeast phase. Lubarskyand P-lunkett (1955) were able to show favorableeffects of increased carbon dioxide on productionof the parasitic phase of C. immitis in complexmedium. All of this evidence agrees with ourobservations that reduced aeration results in anincrease of spherule formation, which may be dueto an accumulation of carbon dioxide in theflask, and that the addition of sodium bicarbonateto the medium can be substituted for reducedaeration with the same results. Additional in-formation on this point is expected from experi-ments being conducted to determine the effectsof continuous passage of an inert gas, containingvarious concentration ratios of oxygen andcarbon dioxide, over cultures incubated inchemically defined medium.

Total growth of cultures was not alwaysaffected similarly by factors enhancing spheruledevelopment. A decrease in total growth alwaysaccompanied an increase in relative spheruleyield due to variation of either the aeration orthe total concentrationof nutrients inthe medium,

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whereas total growth remained constant whenrelative spherule yields were increased by changesin pH, temperature, or reduced illumination.This would seem to rule out adverse growthconditions as a stimulant for in vitro growth ofthe parasitic phase of the organism. It appearsmore likely that inhibition of arthrospore germi-nation, at a specific time during the incubationperiod, is the causative factor.

It was noted that spherules may develop fromsecondary arthrospores following an initialmycelial growth or that they may be converteddirectly from the arthrospores in the inoculum.The number of spherules present in the culturesgreatly exceeds the number of arthrosporesoriginally present in the inoculum, indicatingintermediate mycelial development. Also thelength of spherule chains, shown in figures 10,11, and 12, is far greater than that of the arthro-spore chains introduced inthe inoculum. However,microscopic examinations of cultures incubated at41 C indicated a direct conversion of the ar-throspores in the inoculum to spherules, withcomplete absence of arthrospore germination atthis temperature.

Production of spherules in chemically definedmedium would materially enhance physical,biochemical, and nutritional studies of theparasitic phase of C. immitis. It would alsopermit the accumulation of large amounts ofantigenic material, free from foreigm protein.

ACKNOWLEDGMENTS

The author wishes to thank Henry F. Tantalo,USN, for the valuable technical assistancerendered in this work, and to express appreciationto August E. Selckmann for his time and effortsin making the photomicrographs. Expression ofappreciation is also due Drs. Riley D. House-wright and Edwin P. Lowe for encouragementin this project, and Dr. Norman F. Conant forexaminingculturematerial and photomicrographs.

SUMMARY

Studies were made on some physico-chemicalenvironmental factors affecting the growth of theparasitic phase (spherule) of Coccidioides immitisin a chemically defined liquid medium. Mostsatisfactory production was obtained from young(7- to 14-day) well sporulated inocula, in amedium containing 0.67 per cent solids (total

nutrient content) with initial pH values between6.0 and 8.0, incubated at 37 C in complete dark-ness for 72 hr under reduced aeration.

REFERENCES

BAKER, E. E. AND MRAK, E. M. 1941 Spheruleformation in culture by Coccidioides immitisRixford and Gilchrist. Am. J. Trop. Med.,21, 589-594.

BARNETT, H. L. AND LILLY, V. G. 1950 Nutri-tional and environmental factors influencingasexual sporulation of Choanephora cucurbi-tarium in culture. Phytopathology, 40,80-89.

BULLEN, J. J. 1949 The yeast like form ofCryptococcus farciminosus (Rivolta): (Histo-plasma farciminosum). J. Pathol. Bacteriol.,61, 117-120.

BURKE, R. C. 1951 In vitro cultivation of theparasitic phase of Coccidioides immitis. Proc.Soc. Exptl. Biol. Med., 76, 332-335.

CHRISTENBERRY, G. A. 1938 A study of theeffect of light of various periods and wavelengths on the growth and asexual reproduc-tion of Choanephora cucurbitarium (Berk.and Rav.) Thaxter, J. Elisha Mitchell Sci.Soc., 54, 297-310.

CONANT, N. F. 1941 A cultural study of thelife-cycle of Histoplasma capsulatum Darling1906. J. Bacteriol., 41, 563-580.

CONANT, N. F. AND VOGEL, R. A. 1954 Theparasitic growth phase of Coccidioides immitisin culture. Mycologia, 48, 157-160.

CONVERSE, J. L. 1955 Growth of spherules ofCoccidioides immitis in a chemically definedliquid medium. Proc. Soc. Exptl. Biol. Med.,90, 709-711.

HENRY, W. B. AND ANDERSON, A. L. 1948 Spor-ulation by Piricularia oryzae. Phytopa-thology, 38, 265-278.

LACK, A. R. 1938 Spherule formation and endo-sporulation of the fungus Coccidioides immitisin vitro. Proc. Soc. Exptl. Biol. Med., 38,907-909.

LUBARSKY, R. AND PLUNKETT, 0. A. 1955 Invitro production of the spherule phase ofCoccidioides immitis. J. Bacteriol., 70, 182-186.

MACNEAL, W. J. AND TAYLOR, R. M. 1914Spherule formation in vitro. J. Med. Re-search, 30, 261.

MADER, E. 0. 1943 Some factors inhibiting thefructification and production of the culti-vated mushroom, Agaricus campestris L.Phytopathology, 33, 1134-1145.

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ROESSLER, W. G., HERBBT, E. J., MCCULLOUGH,W. G., MILLS, R. C., AND BREWER, C. R.1946 Studies with Coccidioides immitis.I. Submerged growth in liquid mediums. J.Infectious Diseases, 79, 12-22.

SALVIN, S. B. 1949a Phase-determining factorsin Blastomyces dermatitidis. Mycologia, 41,311-319.

SALVIN, S. B. 1949b Cysteine and related com-pounds in the growth of the yeast-like phase

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YARWOOD, C. E. 1941 Diurnal cycle of ascusmaturation of Taphrina deformans. Am. J.Botany, 28, 355-357.

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