Vol. 57, No. 10

NOTES

Influence of Potassium in the Agar Medium on the Growth Patternof the Filamentous Fungus Fusarium solani

JOACHIM DASBiochemisches Institut der Medizinischen Fakultat, Christian-Albrechts-Universitat, Kiel, Germany

Received 14 February 1991/Accepted 6 July 1991

A decrease in the concentration of K+ ions below 3 mM in agar medium which also contained starch, caseinhydrolysate, MgSO4, and K2HPO4 changed the growth pattern of Fusarium solani illuminated in diurnal 12-hlight/12-h dark cycles from zonation to a feathery growth mode. Rubidium or cesium ions could replacepotassium, but lithium, sodium, and the bivalent alkaline earth ions could not.

When certain fungi are inoculated on an agar plate, a

concentric ring pattern of sporulation or conidiation aroundthe point of inoculation, called zonation, is formed (8). Insome fungi, an endogenous zonation occurs. Examples are

Neurospora crassa, with a circadian rhythm (6, 13), andNectria cinnabarina, with a noncircadian rhythm (1). In a

previous paper (4), we reported on the influence of cyclicillumination on the growth pattern of the filamentous fungusFusarium solani (Mart.) Sacc. In that case, it was notpossible to induce any endogenous rhythm. However, whendiurnal (daily) light-dark (LD) cycles were applied to thefungus, a zonation was found, whereas in continuous light or

darkness zones of conidiation failed to appear. Since then,other factors have been found which can influence thegrowth pattern of F. solani. In this contribution, the effect ofsome nutrients in the growth medium, especially that ofpotassium ions, on the morphogenesis of F. solani on agar

plates is described.Organism and growth conditions. F. solani 62416 was

obtained from the German Collection of Microorganisms,Gottingen, Germany. Standard agar medium was preparedby the method of reference 4: 5 g of soluble starch, 2.5 g ofcasein hydrolysate (Casitone; Difco Laboratories, Detroit,Mich.), 0.5 g of MgSO4 7H20, 0.328 g of K2HPO4- 3H20,and 15 g of agar were dissolved in 1 liter of distilled water.Sometimes the medium was solidified by the addition of1.5% agarose (type NA; Pharmacia, Uppsala, Sweden) or

silica gel (type 60; Merck, Darmstadt, Germany; washedwith chrome sulfuric acid). In some experiments the caseinhydrolysate was replaced by an equal amount (2.5 g/liter) ofNaNO3, and in other experiments K2HPO4 was replaced byamounts of Li2SO4, Na2HPO4, Rb2SO4, Cs2SO4, BeSO4,MgSO4, CaCl2, SrCl2, or BaCl2 which were equimolar withrespect to K+ in K2HPO4. All reagents were of the highestquality available and, if not stated otherwise, were pur-chased from Merck. As a precaution to minimize contami-nation with K+, the stock agar and the glass flasks contain-ing the medium during autoclaving were washed, in mostexperiments, with distilled deionized water at room temper-ature before use. The glass flasks were thrice filled withwater for 0.5 h. The agar (15 g) was incubated for 2 to 6 hwith 1.6 liters of water in a flask with stirring. Then the agarsuspension was filtered on a paper filter (type 595; diameter,55 mm; Schleicher & Schull, Dassel, Germany) and washed

four times with 1 liter of water each time. The efficacy of thewashing step was not tested. The other constituents of themedium were then added to the washed agar.

The medium was autoclaved for 30 min at 120°C andpoured into plastic petri dishes (diameter, 9 cm) to a depth of3 mm, after which the pH of the medium was between 6.8and 7.2. In some experiments, the mold was cultured on

cellulose nitrate microfilter disks (diameter, 7 cm; pore size,0.2 R,m; type 11307, Sartorius, Gottingen, Germany) placedon agar plates (4). Usually, a bundle of macroconidia (105)was centrally inoculated on the surface of an agar plate. Thecultures of F. solani were kept in a cabinet (manufactured byour workshop) in which the temperature was maintained at20°C and the light could be automatically switched on and offin accordance with a chosen program. During the lightphase, the culture dishes were illuminated by six fluorescenttubes (Phillips TLD 15W/25) placed 5 cm above the petridishes. The cyclic LD phases (12 h of light/12 h of darkness)were controlled with a timer (4). Each experiment was

repeated at least 10 times.Flame photometry. The constituents of the standard agar

medium were tested for contamination with potassium byflame photometry at 768 nm, using an Eppendorf flamephotometer (Netheler & Hinz, Hamburg, Germany). Thecalibration of the photometer, using solutions containing 0,0.2, 0.4, 0.6 ... up to 1.0 mM K+, yielded a straight linethrough the origin. The K+ concentration was measured indeionized water solutions of 0.05% MgSO4 7H20, 0.5%starch, 0.25% casein hydrolysate, and 1.5% cold suspendedagar and in a suspension of macroconidia (5 x 105/ml). Theconcentrations of the compounds of the culture mediumwere identical to those in the agar medium used for growth.The water and the solutions of MgSO4, starch, and caseinhydrolysate were autoclaved before measurement. Distilleddeionized water was used as a blank.

Staining. The cultures were stained with Coomassie bril-liant blue R-250 (Serva, Heidelberg, Germany) by a proce-dure used for the staining of proteins in gels (12) to visualizethe colorless mycelia in the agar.

Influence of constituents of the agar medium on growthpattern. F. solani inoculated on an agar plate containingsoluble starch, casein hydrolysate, magnesium sulfate, andpotassium phosphate and grown in an LD cycle of 12 h:12 h

developed a zonation pattern (Fig. 1). When starch, casein

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FIG. 1. Growth pattern (zonation) of F. solani on an agar platecontaining soluble starch, casein hydrolysate, magnesium sulfate,and potassium phosphate at 20'C (complete medium). Macroconidia(10') of F. solani were inoculated in the center of the petri dish. TheLO regimen was 12 h of light followed by 12 h of darkness. Thecolony was stained with Coomassie blue. Bar, 1 cm.

hydrolysate, or MgSO4 was omitted from the agar medium,the fungus grew homogeneously, i.e., without developingzones, in a 12-h:12-h LD cycle (Fig. 2). However, thedensity of mycelial growth differed. This could be detectedby eye when the cultures were compared, especially afterthey were stained with Coomassie blue. The denser growthwas found on agar plates containing all four nutrients. The

FIG. 2. Homogeneous growth of F. solani on an agar plate withcomplete medium lacking magnesium sulfate. Other conditions wereas given in the legend to Fig. 1.

FIG. 3. Change in growth of F. solani from zonation to afeathery pattern on an agar plate containing complete mediumwithout potassium phosphate. Conditions were the same as given inthe legend to Fig. 1.

density was lower than that of the complete nutrient mixtureon media without MgSO4, even lower on media withoutstarch and casein hydrolysate, and lowest on a simple 1.5%agar gel. The growth pattern on agar plates containingstarch, casein hydrolysate, and MgSO4, but no K2HPO4,was peculiar (Fig. 3). Zones were visible in the center of theculture dish. Elsewhere, the mold grew radially, but ramifiedlike the branches of a tree. In the case of Macrophomiaphaseolina, this was called feathery growth (16). Hence, allfour nutrients in the agar medium were necessary in theranges of concentration used to sustain the zonation process.The growth velocity was the same on media lacking any oneof the constituents as on the complete medium, 3 mm/day(4).The nature of the transition from zonation to the feathery

growth pattern on media devoid of potassium phosphate wasexamined more closely. First, we tested whether the potas-sium or the phosphate ion was the factor responsible for thechange in the mode of growth. In some experiments, theK2HPO4 was replaced by an equimolar concentration of KClor MgHPO4. With KCl added to the agar, i.e., when phos-phate was absent, normal zonation appeared. Conversely,when potassium was absent but phosphate (MgHPO4) waspresent, the zonation was replaced by the feathery pattern.Moreover, if inoculated media without potassium were ex-posed to continuous darkness or continuous light, then thezones in the center of the agar plate were replaced by ahomogeneous growth, which is characteristic of growth incontinuous darkness or continuous light (4). However, theperipheral growth on such media was feathery. Thus, in theabsence of potassium, light was not able to induce theformation of zones. Hence, three modes of growth werefound: (i) homogeneous growth in continuous darkness orcontinuous light in the presence of K+ (4); (ii) zonation inLD in the presence of K+ (4); and (iii) feathery growth in

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VOL. 57, 1991

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FIG. 4. Number of zones developed by F. solani on an agar plate(diameter, 9 cm) as a function of the concentrations of potassium(A), rubidium (0), and cesium (OI) in the agar medium (dose-response curve). F. solani was inoculated in the center of each petridish. The cultures were incubated at 20°C in an LD regimen of 12 hof light followed by 12 h of darkness. The dashed line through themeasured values is given for orientation.

continuous darkness, continuous light and LD in the absenceof K+.

Evidently, the phosphate ion did not cause the zonationeffect, although this ion plays an important role in regulationof the growth pattern of filamentous fungi, e.g., Ascochytarabiei, Aspergillus giganteus, Fusarium spp., and Leptos-phaera michotii (2, 8, 11, 19). Instead, the potassium ion wasresponsible for induction of the zone-forming process. In N.crassa, the Na+/K+ ratio also played an important role inmorphogenesis (18).

Influence of ions of uni- and bivalent alkali metals on

growth. The effect on the growth of F. solani of a variety ofions in the medium was tested in a series of experiments.The anions Cl- (3 to 9 mM), PO43- (1 to 4 mM), and S042-(2 to 5 mM) had no influence on the growth pattern. (Theconcentration ranges tested are given in parentheses.) Inother experiments, K+ was replaced by equimolar concen-trations of Li', Na+, Rb+, Cs+, Be2+, Mg2+, Ca2, Sr2+,and Ba2+, whereas the concentrations of the other constit-uents of the growth medium were kept at the levels in thecomplete medium. The bivalent alkaline earth ions and theunivalent alkali metal ions, Li+ and Na+, failed to supportzonation. Only Rb+ and Cs+ were able to substitute for K+in the development of zones. The addition of BeSO4 ren-dered the medium acidic (pH 4.0) and prevented the agarmedium from solidifying after autoclaving. However, itseems unlikely that the higher viscosity of the agar mediumfavored the feathery pattern over the zonation mode.The similarity of the effects of potassium, rubidium, and

cesium ions on zonation may be due to the fact that theseions have almost the same diameter in the unhydrated(-0.12 nm) and the hydrated (-0.33 nm) forms (15). Theions of the other alkali metals, sodium (-0.18 and -0.35 nm)and lithium (-0.24 and -0.38 nm), and those of the bivalentalkaline earth elements are considerably larger (15).Development of zones in the center of an agar plate without

addition of alkali metals. In the center of agar plates contain-ing a medium without a potassium salt supplement, morethan five zones could be detected (Fig. 3 and 4). Apparently,a sufficient concentration of potassium ions was present to

FIG. 5. Growth of F. solani on an agar plate with completemedium lacking potassium. No zones were developed. Growth wasstrongly reduced. Shown is a sample of a mycelium after more than3 weeks of growth. The growth conditions were the same as given inthe legend to Fig. 1.

support the zonation around the point of inoculation. Theseendogenous K+ stores may originate from the conidia orfrom a contamination with K+ in the other substances in thegrowth medium. However, in macroconidia no K+ could bedetected by flame photometry.The second possibility, contamination of the constituents

of the growth medium with potassium salts, was tested withagar plates in which the K+ content had been consumed bya previous culture of F. solani. Conidia of the mold wereinoculated on a microfilter (diameter, 7 cm; pore size, 0.2,m) covering an agar medium which lacked K2HPO4. Thesmall pores prevented the fungus (thickness of the hyphae, 5to 10 ,um) from growing through the filter into the agarmedium. However, the dissolved nutrients could penetratethe pores (4). In a 12-h:12-h LD regimen, the pattern on thefilter was similar to that on uncovered agar. After the fungushad covered the filter, the latter was removed. When theK+-depleted agar plate was directly inoculated with conidiaonce more, i.e., without a filter, no zones were visible, butthe feathery pattern appeared (Fig. 5). Moreover, the radialgrowth had been strongly retarded by this procedure (fromabout 3 mm/day to 5 to 7 mm/week). The result was that themycelia reached, at most, a diameter of 2.5 cm beforegrowth stopped. Other experiments failed to demonstratethe presence of any diffusible waste compounds or growthfactors excreted into the medium which could account forthe observed phenotypes (3).The concentration of the contaminating potassium ions in

the constituents of the agar medium was determined byflame photometry. Cold unwashed agar contained 0.06 mMK+. After autoclaving, K+ could not be detected in distilledwater, washed agar, or a 0.5% starch solution. A 0.05%Mg2SO4 solution and a 0.25% casein hydrolysate solutioncontained 0.02 and 0.13 mM K+, respectively. Rb+ and Cs'have not been determined since the methods required arehighly complicated and since it was considered highly un-

1 10

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likely that these rare elements would be present in significantconcentrations in the reagents used. Hence, most of thecontamination of K+ was introduced into the medium by thecasein hydrolysate and not by the agar used. This agreedwith results obtained with agarose or silica gel plates, on

which the same growth patterns were seen as on normal agarplates. The discrepancy between the overall concentrationof 0.15 mM K+ in the agar medium without added K2HPO4and the cutoff concentration for the development of zones,which is -0.03 mM, may be due to an accumulation of K+ inthe center of the plate above this threshold caused by themold and leading to about six zones (Fig. 4). Subsequently,the K+ concentration left in the remainder of the plate isinsufficient to sustain any further zonation. The resultssupport the assumption that the contamination with K+ was

transferred to the agar plate not by the macroconidia of theinoculum but by the nutrients, especially the casein hydroly-sate. It was not possible to avoid an addition of K+ to theagar medium by casein hydrolysate by using equal amountsof NaNO3 instead of casein hydrolysate since such a replace-ment reduced the growth and only weak zonation could beinduced on such a medium. It is possible that other factors(e.g., vitamins) present in casein hydrolysate (5) also con-

tribute to the zonation process.Dose effect of the concentrations of potassium, rubidium,

and cesium ions on number of zones. Figure 4 shows thedependence of zonation on the concentrations of potassium(K2HPO4), rubidium (Rb2SO4), and cesium (Cs2SO4) in theagar medium. The number of zones developed is plottedversus the logarithm of the concentrations. On media with-out added alkali metal ions, six zones (±1) were observed,and the hyphae grew in the feathery fashion on the remain-der of the agar plates. The addition of an alkali ion concen-

tration of up to 3 x 10-2 mM did not change the pattern.However, above this threshold the number of zones in-creased. Thus, an alkali ion concentration of -3 mM, i.e.,the concentration used in the complete medium, was suffi-cient to sustain a zonation across the agar plate (-15 zones).Therefore, three concentration ranges of K+ may be definedby the action of this ion on the growth of F. solani. At highconcentrations of K+ (.3 mM), zones could be developed,but at very low K+ concentration, the limit of which was notdetermined, no growth was possible. In an intermediateconcentration range of K+, tentatively given as 0.5 to <0.1mM, growth was still possible but no zones could be formed.The mold grew homogeneously.Mechanism of action of the potassium ion. The physiolog-

ical mechanism of action of the K+ ion in zonation is stillunknown. In the filamentous fungus Aspergillus niger, theratio of the concentration of glucose to potassium ionsinfluences the endogenous zonation rhythm (9, 10). In thatorganism, Rb+ could replace K+, but Cs', Na+, and Li'could not. Jerebzoff (9, 10) suggested that K+ affects an

enzyme complex which metabolizes isoleucine. Further-more, K+ influences the osmotic balance in plant and fungalcells (14). It also is involved in membrane processes, e.g., insustaining a potential across a membrane. In the last case, itmust be transported through the cell membrane, eitherpassively through ion channels (7) or actively by a pump

(17). Usually, channels or pumps can transport only ions ofa certain diameter. The assumption that such membraneprocesses are involved in the development of the growthpattern of F. solani is supported by the results on ionreplacement, in which ions of the same diameter couldreplace each other. However, other mechanisms also mayoperate.

The continuous interest and support of H.-G. Busse and B. H.Havsteen are gratefully acknowledged. Thanks are due to A. Baade,H.-P. Beuck, P. Heyer, S. Ihle, and I. Muller for excellent technicalassistance.

REFERENCES

1. Bourett, J. A., R. G. Lincoln, and B. H. Carpenter. 1971.Modification of the period of a noncircadian rhythm in Nectriacinnabarina. Plant Physiol. 47:682-684.

2. Brown, W. 1925. Studies in the genus Fusarium. II. An analysisof factors which determine the growth form of certain strains.Ann. Bot. 39:373-408.

3. Das, J., and H.-P. Beuck. Unpublished data.4. Das, J., and H.-G. Busse. 1990. Light driven diurnal zonation in

the filamentous fungus Fusarium solani. Int. J. Dev. Biol.34:319-322.

5. Difco Laboratories (Detroit, Mich.). Personal communication.6. Edmunds, L. N., Jr. 1984. Physiology of circadian rhythms in

micro-organisms. Adv. Microbiol. R25:61-303.7. Gustin, M. C., B. Martinac, Y. Saimi, M. R. Culbertson, and C.

Kung. 1986. Ion channels in yeast. Science 59:1195-1197.8. Jerebzoff, S. 1965. Growth rhythms, p. 625-645. In G. S.

Ainsworth and A. S. Sussmann (ed.), The fungi, vol. 1. Aca-demic Press, Inc., New York.

9. Jerebzoff, S. 1976. Metabolic steps involved in periodicity.Dahlem Konferenzen. Life Sci. Res. Rep. 1:193-213.

10. Jerebzoff, S. 1979. System6s m6taboliques oscillants chez lesv6g&taux inferieurs. Bull. Soc. Bot. Fr. 120:23-38.

11. Jerebzoff-Quintin, S., and S. Jerebzoff. 1981. Cyclic AMP levelin relation to different conditions of sporulation rhythm regula-tion in Leptosphaeria michotii (West.) Sacc. Arch. Microbiol.128:325-329.

12. Klose, J., and M. Feller. 1981. Two-dimensional electrophoresisof membrane and cytosol proteins of mouse liver and brain.Electrophoresis 2:12-24.

13. Lakin-Thomas, P. L., G. G. Cote, and S. Brody. 1990. Circadianrhythms in Neurospora crassa: biochemistry and genetics. Crit.Rev. Microbiol. 17:365-416.

14. Mohr, H., and P. Schopfer. 1978. Lehrbuch der Pflanzenphysi-ologie, 3rd ed., p. 114-134, Springer-Verlag, Berlin.

15. Nightingale, E. R. 1959. Phenomenological theory of ion solva-tion. Effective radii of hydrated ions. J. Phys. Chem. 63:1381-1387.

16. Pearson, C. A. S., J. F. Leslie, and F. W. Schwenk. 1986.Variable chlorate resistance in Macrophomina phaseolina fromcorn, soybean, and soil. Phytopathology 76:646-649.

17. Rodriguez-Navarro, A., M. R. Blatt, and C. L. Slayman. 1986. Apotassium proton symport in Neurospora crassa. J. Gen. Phys-iol. 87:649-674.

18. Sato, R., T. Kondo, and Y. Miyoshi. 1985. Circadian rhythms ofpotassium and sodium contents in the growing front of Neu-rospora crassa. Plant Cell Physiol. 24:447-454.

19. Zurzycka, A., S. Jerebzoff-Quintin, and S. Jerebzoff. 1983.Cyclic AMP phosphodiesterase activity and cyclic AMP levelduring the photostimulated morphogenesis in Aspergillus gigan-teus Wehm. mut. alba Zurz. Arch. Microbiol. 136:199-202.

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