Population density and yeast mycelial dimorphism in Aureobasidium pullulans

[ 39 ] Trans . Br . my col. Soc. 82. ( 1) 39-44 ( 1984)

Printed in Great Britain

POPULATION DENSITY AND YEAST MYCELIAL DIMORPHISM IN AUREOBASIDIUM PULLULANS By DAVID PARK Botany Department, The Queen's University, Belfast BT7 INN At low population densities of A. pullulans, development is mycelial, but above a threshold density of cells there is a quantitative relation between population density and tendency to yeast development. This effect is interpreted as being autogenic. With ammonium nitrogen source there is a lower response of cells to this autogenic influence than with nitrate source. A parallel is drawn with similar relationships in the conidiation of mycelial fungi. The autogenic influence operates in aqueous medium and is transmissible through Cellophane, but no evidence is found for a volatile effect. Ramos & Garcia Acha (1975) obtained different results with two different levels of inoculum density of A. pullulans (de Bary)Arnaud in liquid cultures. With yeast cell, large cell, septate large cell or chlamydospore inoculum material an inoculum density of 1'3 x 105cells em-ain fresh medium gave a budding yeast (Y) growth phase. With the last three types of cell as inoculum material at 6'7 x 10' cells em-a germ-tube production occurred followed by the development of mycelium (M) . Park (1982) has shown that a yeast-cell inoculum at low density can also give germ-tubes and M phase. Brown ( 1965) and Hsiao (1970) have reported that some , but not all, isolates of A. pullulans gave M with ammonium nitrogen source and Y with nitrate nitrogen source . Their level of inoculation was not specified precisely, but the loopful taken from a 3-day culture could have given a low inoculum density in isolates with a low growth rate. The isolate used in the present work would have given an inoculum of 1'5 x 102 cells em-a. The actual inoculum level would be variable with isolates having different growth rates, and this could, as seen from the results reported here, explain the variability in response between their different isolates. Sevilla et al. (1977) failed to confirm this inorganic nitrogen source effect , but they used an inoculum level of 0·01 mg dry weight of organism em-a, which calculations show to be in the region of 106 cells cm". Thus they are likely to have been working above the threshold where the effect could be detected. All the work reported above used long-term assays incubated for several days . A re-examination of the effect of population density on the Y-M response of A. pullulans has been made using the distinction between effects on direct development

where the immediate germination response of inoculum cells is recorded at 7 h, and effects on indirect development from 24 h assays where small colonies have developed (Park, 1982). MATERIALS AND METHODS

Inocula of exponential phase yeast cells of isolate Ai (Park, 1982) (ATCC 48168 ) of A ipullulans were grown in 25 crrr' lots of Brown's (1965) nitrate liquid medium in 100 em" flasks orbitally shaken at 20°C for 2 or 3 days . Cell population densities were determined by haemocytorneter. The germination and growth medium used had the composition: glucose, 0'7 g; MgS0 4· 7H20 , 0'5 g; KH2P04, 0'2 g ; nitrogen source at 0'035 g N « N H4)2S0 4' 0'17 g, or NaNO a, 0'2 g); agar, 10'0 g ; distilled water to 1 1. Agar plates were poured 3 days before use and inoculated each with 0'2 ern" cell suspension distributed over the plate with a bent glass spreader. Where cells were incorporated in agar, molten agar (10 ern") at 40°C was seeded with inoculum cells and immediately either poured into a Petri dish or poured over a sterile glass slide for the thin agar film method (Park, 1983). Cultures were incubated at 25°. RESUL TS

Effect of inoculum density on Y -M dimorphism Figure 1 summarizes data from five different runs for cells spread on nitrate agar at different densities. For direct comparison, and because different batches of inoculum cells do not give the same highest count at low population densities, the values have been normalized by recording each point as percentage maximum M level in the run. Cell population density, initially calculated as cells

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Dimorphism in Aureobasidium

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Fig . 1. D irect mycelial development (7 h assay) and cell population density on spread agar plates : five experiments each represented by a different symbol. Each value is th e mean of three replicate counts of 100 inoculum cells. Fitted linear regression equation for first 13 points : Y = 0'45X -12 '55 (t = 5'873 for 11 D.F. ; P < 0 '001). Fitted linear regression equation for last 12 points : Y = 94 '2 - 8 X 10- 8 X . Dotted lines show 95 % confidence zone for regression slope.

cm", has been converted to mean distance between cells (proport ional to square root of the inverse of population density), as being the most appropriate scale for comparisons. The calculated linea r regression of best fit of perc entage M on distance between cells for the first 13 points on the graph gives a slope that meets the horizontal plateau at a

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Effect of inoculum density on indirect development The effect of population density on indirect development, i.e. on colony form (M or Y) at 24 h, has also been exam ined. Fig . 3 show s the results of

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distance just over 200 pm. Thus at mean distances less than this th e cells influence each other to increase the Y tendency. At greater mean distances (at this time of observation) the maximum M tendency reveals itself with no apparent cell proximity effect . Other observations have been made on cells seeded in nitrate agar . The thin agar film assay (Park, 1983) was used for two observations and cells seeded in agar poured as plate s for a third observation (Fi g. 2). The relationship between mean cell proximity and percentage maximum mycelium is similar in form to that for cells spread on agar, but the proximity effect increasing the Y tendency operates over greater distances here. Maximum M tendency is attained here at mean distances above 450 pm, which is more than double the effect ive mean distan ce observed in surface spreads. This may be due to the absence of a large source/sink below the cells such as occurs in the surface spread design. Over a range of high population den sities there is in both situ ations a clear quantitative relationship between cell proximit y and the shift from M development to Y development.

~ 0 100 200

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Mean distance between cells (/l m) Fig . 2. Direct mycelial development (7 h assay) and population density of cells seeded in agar : circles for 10 em" agar in Petri dishes, squares for two experiments using thin agar film assay on glass slides. Each value is the mean of three replicate counts of 100 inoculum cells. Fitted linear regre ssion equation for points nearer than 450 pm : Y =0'25X-13 '02 (t= 6'992 for 17 D.F. ; P < 0 '001). Dotted lines show 95 % confidence zone for regression slope.

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Fig. 3. Indirect mycelial development (24 h assay) and population density of inoculum cells spread on agar: two experiments (0, e ). Each value is the mean of three replicate counts of 100 colon ies. Fi tted linear regression equation for the first ten points : Y = 108 '4X-20'1 (r = 7'501 for 8 D.F.; P < 0 '001 ). Dotted lines show 95 % confidence zone for regression slope .

D. Park Table 1. Percentage of initial direct mycelial development (7 h) persisting as mycelial colonies at 24 h: values calculated from tables I and ], Park (19 82)

Nitrate

No nitrogen Ammonium 55'3

two experiments over slightly different population density ranges. Maximum mycelial persistence in colonies is achieved in this assay only at inter-colony distances of about 1 mm and greater. Colonies in spreads with a mean distance less than this show a quantitative relationship between proximity and the shift of development from M to Y. This effect is readily understood as an extension of that observed in the previous two experiments if individual cell-eell effects also operate among groups of cells. The greater effectiveness with distance probably reflects the increased biomass present after the further 17 h growth which allows for about seven further mass doubling times under these conditions. This effect can be appreciated from Table 1, calculated from the results of Park (1982, tables 1, 3), which shows that for different media the M values recorded at 24 h are reduced from those for initial direct development. In this table if the type of development remained unchanged, values of 100% would result. The calculations support the present comparison showing that the tendency to Y development increases with continued incubation beyond 7 h. Effect of population density/nitrogen source interaction on development The reference to Table 1 together with the findings reported here raise a question about the conclusion

41

arrived at by Park (1982), that while the type of inorganic nitrogen source influenced indirect development at 24 h it had no influence upon the form of direct development of A. pullulans cells on initial germination. That conclusion was at variance with earlier statements in the literature (Brown, 1963; Ramos & Garcia Acha, 1975). The question is whether there exists an interaction between cell population density and any influence of type of nitrogen source such that at high population densities ammonium nitrogen source has a greater effect than nitrate nitrogen source in relieving the cell-eell proximity effect causing the shift from M to Y development of germinating cells. If so, then such influence may have been missed by Park (1982), who chose to use low population densities that were outside the proximity range shown in the present work for the shift. An experiment was done to examine the effects of ammonium and nitrate on direct development at a range of population densities in agar spreads (Table 2). It is evident from the statistical analysis that for level of both Y development and M development there is no significant interaction between type of nitrogen source and population density, and also that there is no significant difference between the two nitrogen sources across the range of population densities. The only significant differences are those resulting from the effects of population density. This confirms and extends the results shown in Fig. 1. Thus Park's (1982) conclusion that the type of inorganic nitrogen source does not influence the form of direct development is supported over a range of population densities including both those where the autogenic density effect reverses the normal tendency to M development and those where it does not. For indirect development, on the other hand, Park (1982) observed that the type of nitrogen

Table 2. Direct development (7 h assay) and cell population density/nitrogen source interaction. Each value is the mean of three separate counts of 100 inoculum cells Cells

Mean cell proximity

M%

y%

(cm ") (pm) NH4 N03 N H4 N04 2,82x 10' 67'2 22'00 18'33 24'67 22'33 1'41 x 10' 16'00 20'00 95'0 19'00 27'67 7'05 x 103 134'4 33'67 10'33 34'33 17'33 3 3'52 x 10 190'2 8'00 11'00 42'33 43' 67 3 l'76x 10 269'0 10'00 52'67 48'67 7'33 8'81 x 102 380'2 61'67 6'00 44'33 7'33 Two-way Anova (with arcsin transformation): F ratios for M%: Cell density = 27'52 (5/5 D.P.); P
N.S. N.S,

Dimorphism in Aureobasidium

42

the quantitative value of the population density effect. This is in agreement with the earlier statement (Park, 1982) that 'the M -+ Y transition is autogenic rather than being a direct result of an environmental trigger, but may be influenced in rate by environmental features includin g nitrogen source ' .

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Mean distance between colonies (urn) Fig . 4. Indirect mycelial development (24 h assay) and population dens ity of inoculum cells spread on agar with different nitrogen sources: e, NH.; 0, N03 • Each point is the mean value for three replicate counts of 100 colonies . Fitted linear regression equations for the first five densities: NH,; Y = 0'041X +48'55 (t = 7'55 for 13 D.F. ; P <0"001 ) : N03 ; Y = 0'100X - 24'75 (r = 15'67 for 13 V.F. ; P < 0'001). Dotted lines show 95 % confidence zones for the regression slopes . Two-way Anova on total data (with arcsin transformation ) gives F ratios for : population density = 8·80 (5/ 5 V.F. ), P < 0'05; nitrogen source = 317 '43 (1/ 24 D.F. ), P < 0'001 ; interaction = 23'54 (5/24 D.F .), P < 0 '01.

source did affect the outcome. As observed in 24 h assay here, it is seen (F ig. 4) that the effect of ammonium in supporting a greater M persistence as against nitrate occurs only with population densities high enough to cause some shift in normal M development. Where population density is below the threshold at which an autogenic effect causes a shift from M development there is no difference between the two nitrogen sources. Further, the threshold for this effect appears to occur at the same population density for both nitrogen sources; both calculated linear regression slopes intersect 100 % M development at a mean distance of 1240 p.m. Only the slope of the regression of M development on population density is affected by the type of nitrogen source; that is, the effect of nitrogen source is to influence

Auto- and allo-induced Y development So far the proximity effect has been referred to in terms of its operation within a single population. It may be shown further that one population may affect another population nearby. If an 8 em diam disk of sterile Cellophane PT no . 300 is laid over an established circular colony of A. pullulans growing on agar from a central inoculum, and fresh inoculum cells are spread over the Cellophane at a density that would normally permit mostly M development then after incubation for 7 h this is what occurs where the Cellophane covers uncolonized agar, but over the established colony any development of the added cells is always as Y. Similarly if a colony of A. pullulans growing on a Cellophane disk is lifted with the Cellophane from its agar medium and laid over another established colony on another plate, then any part of its mycelial margin that comes to lie over uncolonized agar continues its mycelial development by apical hyphal growth, but any part of the margin that comes to lie over an older part of the colony below undergoes an immediate change, Firstly, apical growth ceases, then the hyphae become divided by septa into short compartments up to the apex, The hyphae then bud yeast cells laterally and from their apices. Thus an established differentiated colony already showing the M -+ Y shift within itself is able to induce the sh ift in overlying mature marginal hyphae as well as maintain the condition in overlying Y cells, and the influence is transmissible through Cellophane film. During data recording in 24 h assays an uneven distribution of percentage M colonies within surface spread plates has been observed. Table ' 3 shows data from two observations comparing counts in a central 5 em diam area of plates with those in the peripheral 1'0 em wide zone. Colonies

Table 3. Edge effect on mycelial persistence at 24 h Mean

% M colonies at 24 h

Inoculum level Cel1s

(pm)

cm "

3'07 3'78

X X

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9 6'49 124"29

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Edge : 1 em

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D. Park

43

developing near the edge of the dish have a higher among yeasts, and these parallel some of the probability of retaining the M phase for longer. mechanisms of conidial ontogeny in mycelial fungi Since this might have been the result of loss from (von An, 1980). Also conidiation occurs in the internal atmosphere through the gap between mycelial fungi after a period of mycelial growth and base and lid of a volatile material promoting the in regions where biomass build-up in the medium M -+ Y shift attempts have been made to detect has occurred. The influence there may be transsuch an effect. Growing populations of A. pullulans mitted from a grown, conidially competent colony, cells in nutrient medium have been placed in one to young, precompetent, somatic hyphal tips basal halfof a Petri dish and test cells spread on agar through a Cellophane membrane (Park, 1963). The arranged in another basal half dish, the two bases present finding on the ammonium/nitrate influence being fitted together to minimize exchange between on the autogenic development of the Y phase in A. internal and external atmospheres. In no experiment pullulans parallels similar effects of ammonium as of this son has any significant difference been against nitrate in delaying conidiation in fungi such detected between cells given such treatment and as Neurospora crassa Shear & Dodge (Weiss & control cells with distilled water or uninoculated Turian, 1966), Aspergillus niger van Tieghem medium across the internal gap. There is no (Smith & Galbraith, 1971) and Trichoderma evidence that any volatile factor causes the harzianum Rifai (Zuber & Turian, 1981). The implication from the present experimental results autogenic M -+ Y shift. (Fig. 4) is that the influence of inorganic nitrogen source is not on the mechanism producing the cell DISCUSSION proximity effect controlling the shift M -+ Y but on Promotion of Y phase by high population density the tolerance of the cells, or their quantitative has been reported in other Y-M dimorphic fungi: response, to that mechanism where it operates. Candida albicans (Robin) Berkout (Odds, Hall & This interpretation also fits with the lack of Abbott, 1978), Ceratocystis ulmi (Buism.) C. influence of exogenous nitrogen source on the Moreau (Kulkarni & Nickerson, 1981), Histoplasma initial response, where cells have all been pre-grown capsula tum Darling (Scherr, 1957), Mucor rouxii in standard (nitrate) medium, and where response (Calmette)Wehmer (Bartnicki-Garcia & Nickerson, may be expected to be determined by an adequate 1962), Mycotypha spp. (Hall & Kolankaya, 1974; endogenous condition. A working hypothesis for Y-M dimorphism in Schultz, Kraepeling & Hinkelmann, 1974), Vertici//ium albo-atrum Reinke & Berthold (Keen, A. pullulans is suggested whereby the M phase is Wang, Long & Erwin, 1971). In some of these the normal somatic growth phase. This is the form papers it is suggested that there could be a role for that is produced predominantly when yeast or other self-produced mycelial inhibitors, depletion of cells (large cells, septate large cells, chlamydospores) nutrients essential for the development of M phase, germinate at low population densities under or autocatalytic production of factors essential for adequate nutrition. The Y phase then may be the development of Y phase, but no firm evidence regarded as a conidiating phase that can undergo for any mechanism has yet been presented for any microcycle conidiation under some conditions that dimorphic fungus. Lingappa & Lingappa (1969) are more restrictive than those permitting mycelial working with Glomerella cingulata (Stonem.) growth. This type of conidiation has been shown Spauld. & Schrenk reponed that a yeast-like for other fungi that regularly inhabitthephylloplane growth phase develops at high inoculum density. (Dickinson & Bottomley, 1980; Skidmore, 1976), This phase is also described as 'conidial', the and may have some selective advantage in that conidia being 'capable of germinating into second- habitat under conditions of local crowding or ary conidia with an abridged or a lack of nutrient limitation. There is no evidence that the development of mycelial phase'. This can be proximity effect is produced as the result of a regarded as microcycle conidiation. Lingappa & volatile factor via the atmosphere, but the influence Lingappa considered that diffusible substances does operate through aqueous media (including associated with the conidia and not found in the agar medium) and is transmissible through a mycelium were responsible for the inhibition of Cellophane membrane. There is nothing in any of mycelial development and preferential development the data available to suggest that the effect is caused of this Y phase in G. cingulata. by production and excretion into the medium of The comparison of development of the Y phase any substance by the metabolizing cells; it may with conidiation in mycelial fungi is of interest. In equally well result from removal by the cells of G. cingulata the phenomenon appears (Lingappa & some substance required by the fungus at a Lingappa, 1969) to be the same. Different particular level to maintain the M phase. The edge ontogenetic mechanisms of budding are found effect in spread plates in 24 h assays is probably best

Dimorphism in Aureobasidium

44

explained on the difficulty of spreading cells uniformly right up to the agar edge, thus a source/sink of marginal agar is left uncolonized causing a less marked effect in the medium in that region.

REFERENCES

ARX, J. A. VON (1980). A mycologist's view of yeasts. In Biology and Activities of Yeasts (9th Symposium of the Society for Applied Biology), (ed. F. A. Skinner, S. M. Passmore & R R Davenport), pp. 53--61. London, U.K.: Academic Press. BARTNICKI-GARCIA, S. & NICKERSON, W. J. (1962). Nutrition, growth and morphogenesis of Mucor rouxii. Journal of Bacteriology 84, 841-858. BROWN, R. G. (1965). Biochemistry of morphogenesis in black yeastlike fungi. Ph.D. Thesis, Rutger's, The State University, New Brunswick, N. Jersey, U.S.A. DICKINSON, C. H. & BOTTOMLEY, D. (1980). Germination and growth of Alternaria and Cladosporium in relation to their activity in the phylloplane. Transactions of the British Mycological Society 74, 309-319. HALL, M. J. & KOLANKAYA, M. (1974). The physiology of mould-yeast dimorphism in the genus Mycotypha (Mucorales). Journal ofGeneral Microbiology 82, 25-34. HSIAO, M. M. C. (1970). Effect of inorganic nitrogen sources on growth and cell wall composition in black yeastlike fungi. M.Sc. Thesis, Dalhousie University, Nova Scotia, Canada. KEEN, N. T., WANG, M. C., LONG, M. & ERWIN, D. C. (1971). Dimorphism in Verticillium albo-atrum as affected by initial spore concentrations and antisporulant chemicals. Phytopathology 61, 1266-1269. KULKARNI,R K.&NICKERSON,K. W.(1981).Nutritional control of dimorphism in Ceratocystisulmi, Experimental Mycology 5, 148-154. LINGAPPA, B. T. & LINGAPPA, Y. (1969). Role of auto-inhibitors on mycelial growth and dimorphism of Glomerella cingulata. Journal of General Microbiology 56,35-45. ODDS, F. C., HALL, C. A. & ABBOTT, A. B. (1978).

Peptones and mycological reproducibility. Sabouraudia 16, 237-246. PARK, D. (1963). Evidence for a common fungal growth regulator. Transactions of the British Mycological Society 46, 541-548. PARK, D. (1982). Inorganic nitrogen nutrition and yeast-mycelial dimorphism in Aureobasidium pullulans. Transactions of the British Mycological Society 78, 385-388. PARK, D. (1983). Assay systems for the study of yeast-mycelial dimorphism. Transactions of the British Mycological Society 81, 168-172. RAMos, S. & GARCiA ACHA, I. (1975). A vegetative cycle of Pullularia pullulans. Transactions of the British Mycological Society 64, 129-135. SCHERR, G. H. (1957). Studies on the dimorphism of Histoplasma capsulatum. I. The role of -SH groups and incubation temperatures. Experimental Cell Research 12,92- 10 7. SCHULTZ, B. E., KRAEPELING, N. & HINKELMANN, W. (1974). Factors affecting dimorphism in Mycotypha (Mucorales): a correlation with the fermentation/respiration equilibrium. Journal of General Microbiology 82, 1-13. SEVILLA, M. J., ISUSI, P., GUTIERREZ, R, EGEA, L. & URUBURU, F. (1977). Influence of carbon and nitrogen sources on the morphology of Pullularia pullulans. Transactions of the British Mycological Society 68, 300-3°3· SKIDMORE, A. M. (1976). Secondary spore production amongst phylloplane fungi. Transactions of the British Mycological Society 66, 161-163. SMITH, J. E. & GALBRAITH, J. C. (1971). Biochemical and physiological aspects of differentiation in the fungi. Advances in Microbial Physiology 5,45-134. WEISS, B. & TURIAN, G. (1966). A study of conidiation in Neurospora crassa. Journal of General Microbiology 44, 407-4 18. ZUBER, J. & TURIAN, G. (1981). Induction of premature phialoconidiogenesis on germinated conidia of Trichoderma harzianum. Transactions of the British Mycological Society 76, 433-440.

(Received for publication 16 March 1983)