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Development of somatic hyphae of Geotrichum candidum
[ 159 ] Trans. Br. mycol. Soc. 74 (1) 159-165 (1980)
Printed in Great Britain
DEVELOPMENT OF SOMATIC HYPHAE OF GEOTRICHUM CA NDIDUM By
J. M . SMITH
A ND
P. M . ROBI NSO N
Department of B otany , The Queen's University, B elfast BT7 I N N , Northern Ir eland
H ypha e of Geotrichum cand idum Lk ex Pers. ha ve sim ilar ext ensio n rate s and cell dimension s when gro wn either in steady-s tate glu cose-limited ch ernostat cult ure or on solid med ia with low initial glu cose conce ntrations. In both method s of culture th e mycelium is undifferent iated and hyphal development is inco mplete. In liquid media wh ich permit full hyphal development there is a recurrent cycle of development in whi ch each apical cell branches d ichotom ou sly when five or more non ap ical cells have been pr oduced . During each cycle th ere is a grad ual increase in diameter and volume of the api cal cell and each successive non-ap ical cell produced is wider and of greater volume. The mycelium is undifferentiated. The effect of temperature on cell dimensions and hyphaI development is discussed for chemostat culture, batch liquid culture and culture on solid media. In most case s an increase in temperature is associat ed with a decrease in h yphal diameter and cell volume. In an earlier communi cation (Robinson & Smith, 1979) th e development of Geotrichum candid um Lk ex Pers. was described for glucose-limite d che most at culture and for cult ure on soli d med ia. In both cultur e con di tic ns the apica l an d non-apical cells increase d in diam eter and volume as th e speci fic growth rate of th e culture inc reased . These trends were integra ted within a d istinct pattern of hyph al development and, depending on th e con ditions of culture, mycelial differentiation . The present in vestigation involves a m ore detailed comparison of hyph al development of G. candidum in chemostat culture, on solid media and in batch liquid culture. The effect of temperature on hyph aI development is also consi dered . MAT ERIALS AN D M E TH O D S
The same strain of G. candidum used in earlier stud ies (Robin son & Smith, 1976, 1979) was maintained on slopes of Oxoid malt extract agar at 25 °C. For chemostat culture the art hrospores were washed from slopes into the following med ium [g(181) - 1] : D-glucose, 9'0 ; KH 2P04, 3'4; N a2 HP04. 12H20, 8'9; (N H 4)2 S04, 6'0; MgS04 . 7 H20 , 0'2 5; CaCI 2, 0'05 ; ZnS04 . 7H 20, 0'2 ; MnS04 • 7H 2 0 , 0'02 ; CuS0 4 • 5H 2 0, 0'005 ; F eSO•. 7H20, 0'1 ; Na2S04, 0'5 ; Na M 004.2H20 , 0 '005 ; disod ium ethy lenediami ne tetraacetic acid (EDT A), 0 ·6. The glu cose solutio n was ste rilized separately from th e rest of the medium. The chem ostat was a modular fermenter (A. E. G allenk amp & Co. Ltd, T echnico House, London , En gland ) which was modified for use with filamentou s fun gi and
which had a working volume of 0'5 I. The glu coselim ited cul ture was maintained at pH 6'0 ± 0' 1 by th e automatic addition of 0'1 M- NaOH. The culture was agi tated by an impeller and the temperature was controlled at 25 or 30 .L 0'5°. The air flow through the culture was ma intained at 0·6 I m in- I. Solid medi a were prepared by the additio n of Oxoid NO. 1 agar (10 g 1-1) to the same medium as used in th e chernos tat . The level of gluc ose was varied as required. For batch culture in liquid media, aliquots (25 ml ) of chemos tat med ium with a glucose con centration of 10 g I - I were steri lized in 500 ml conical flasks. The med ia were inoc ulated to give an art hrospo re concentra tio n of 10 6 art hros pores rnl" '. H yphal and cell dimensions were determ ined from enl arg ed pr int s of photogr aphs. Straight lines were fitt ed by regression analysis to data in Figs 2, 4 and 5· RESULTS
Correlation of cell dimensions in differ ent grow th conditions
In earlier experime nts (Robinson & Sm ith , 1979) neither mycelial differentiation nor complete hyphal development was detected in gluc oselim ited chemostat cultures of G. candidum under steady-state condi tio ns. Since steady-state cond ition s were establishe d over a relatively narrow range of low res idu al glu cose con centrations, an y parallels in cell dimensions , h yphaI development and growth kinetics m ight be expe cted between chemos tat cultu res and cult ures on solid me d ia
00°7-1536/80'2828-5670 $01.00 © 1980 T he British Mycological Society
Development of G. candidum
160
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Hyphal extension rate (Mill h-1 ) Fig. 1. Cell dimensionsand hyphal extension rates of G. candidum at 30° in chemostat culture and on solid media. Open symbols represent data from chemostat culture; closed symbols represent data from culture on solid media. Apical cells (e,O), non-apical cells (_,0). Each value is the mean of determinations on 20 cells. Vertical lines through symbols represent 95 % confidence intervals where these exceed the height of the symbols.
which contain low initial concentrations of glucose. The cell dimensions recorded for mycelial fragments from chemostat cultures and for colonies on solid media were almost identical for any particular hyphal extension rate which was common to each method of culture. For example (Fig. 1, Table 1), there was good agreement between the dimensions of cells from cultures grown at 30° at a specific growth rate of 0'25 h- I in the chemostat and at an initial glucose concentration (so) of 10 mg I-Ion solid media, and from cultures grown at a specific growth rate of 0'40 h- I in the chemostat and at an initial glucose concentration of 25 mg I-Ion solid media. Similar correlations occurred at 25° (Table 1) between the dimensions of cells from cultures grown at a specific growth rate of 0'30 h- I in the chemostat and at an initial glucose concentration of 10 mg I-I on solid media. For each comparison the hyphal diameters, apical cell volumes, and subapical cell lengths and volumes, were not significantly different. The number of comparisons was limited by steady-state conditions only being possible in the chemostat over a narrow range of residual glucose concentrations and by the difficulty of achieving very low concentrations of carbon source in solid media.
In each comparison in Table 1 the colony radial growth rate (K r ) , which was determined by direct measurement over a period of seven days on solid media, agrees closely with the mean hyphal extension rate (E) for the corresponding chemostat culture. E was determined from the equation E
=
G«,
where G = length of the hyphal growth unit and a = specific growth rate. This equation was established by Steele & Trinci (1975) for undifferentiated mycelia of Neurospora crassa growing exponentially on glucose-limited solid media. The mycelium of G. candidum is undifferentiated in steady-state chemostat culture and the length of the hyphal growth unit varies with specific growth rate (Robinson & Smith, 1979). The appropriate value of the hyphal growth unit for each specific growth rate was substituted in the above equation to give the mean hyphal extension rates for the chemostat cultures. For colonies on solid media the specific growth rate of the main hyphae was calculated from the equation K, = wa, where w = the width of the peripheral growth zone (Trinci, 1971). The width of the peripheral growth zone was assumed equal to the length of
J. M.
Smith and P. M. Robinson
the hyphal growth unit, which was calculated as 150 !Lm from data published by Trinci (1974) for undifferentiated mycelia of G. candidum. Each calculated value for the specific growth rate of the cultures on solid media was almost identical to the specific growth rate for the chemostat culture at which comparable cell dimensions were obtained. The results indicate a similarity in cell dimensions and hyphal extension rate when mycelia of G. candidum are grown in steady-state conditions in a chemostat or on solid media at very low initial glucose concentrations. Under both conditions there is no significant difference between the cell dimensions of main and lateral hyphae, and the cultures can be regarded as undifferentiated (Robinson & Smith, 1979). Development in batch liquid culture Development of somatic hyphae was studied for cultures of G. candidum during growth for 15 h at 30° in a static liquid medium which permitted full hyphal development. The arthrospore density after inoculation was 10 6 arthrospores ml- I • Each germinating arthrospore produced a single main hypha and the apical cell increased in diameter and volume as hyphallength increased (Fig. 2a). Successive non-apical cells were also wider and of greater volume (Fig. 2b) but no trends were
Table
1.
161
detected in the length of either apical or nonapical cells as hyphal length increased. Each apical cell had the capacity to branch dichotomously at the time that five or more (usually eight) septa had been delimited and the majority of hyphae had not fragmented at this stage. The average length of the hyphal filaments was determined at selected times prior to apical branch formation (Fig. 3) and the results indicated that the extension rate of the apical cells increased throughout this phase of development. In cultures incubated at 30° there was no difference in non-apical cell dimensions, in filament length at the time of formation of the first apical branch, and in the number of septa at this time, between hyphal filaments resulting from an initial arthrospore concentration of either 10 4 or 10 6 arthrospores ml- I (Table 2). Numerous mycelial fragments were present in cultures which had been incubated for 24 h. Each fragment had the potential to branch apically and there was a progressive increase in the diameter and volume of apical cells and of the sequence of non-apical cells formed prior to apical branch formation (Fig. 4a). There was no trend in cell length. Immediately after apical branch formation the first subapical cell formed by each new apical cell was narrow and of low volume. Each new apical cell, and the succession of non-apical cells
Cell dimensions and growth rates of hyphae of G. candidum in continuous culture and on solid media 30"C
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Hyphal width (/lm) 3,87=°'16* Apical cell length (/lm) 152::,,8'8 Apical cell volume (/lm 3) 1788_: 158 Subapical cell length (/lm) 53'OL 3'3 Subapical cell volume (/lm 3) 626 : 68
Meanhyphal extension rate (/lm h- I) Specific growth rate (h- I)
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MYC
74
Development of G. candidum
162 Table
2.
Hyphal development and average dimensions of non apical cells of G. candidum in static liquid culture
Spore density (arthrospores Temperature Cell length (aC) ml- 1) (pm) 20 2'78 x 10' 129'4 ± 13'9* 2'78 x 1O' 30 73'8 ± 7'3 2'78 x 104 66'o ± 6'3 3°
Cell diam (pm)
Cell volume (pm 3)
4'83 ±o'23 4'43 ±O '18 4'53 ±O'16
2371 ± 219 1149±1 27 1064± 88
Number of septa in filament 8'18 ±l '03 T82 ±O'99 9'12 ±1 '64
Length of filament (lim) 1230 ± 105 672 ± 52 611 ± 128
*
95% confidence intervals. Each result is the mean of determination s on 11 hyphal filaments, each of which had produced a single apical branch .
which were produced, showed the same increase in diameter and volume already described. This recurrent cycle of development was a constant feature of all hy phal fragments and could be identified in hyphae which formed eit her as lateral branches or as ap ical branches (F ig. 4b).
Effect of temperature on development of cells and hyphae In chemostat culture a shift in temperature from 25 to 30° results in several developmental changes
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for a given specific growth rate. The diameter of apical and subapical cells decreases, as does the volume of the apical cells . Apical and subapical cells are longer, and fewer cells form lateral branches (Robin son & Smith, 1979). The dimensions of apical and subapical cells of G. candidum on solid media were also affected by temperature. For a given temperature, the lengths of the apical cells did not differ significantly throughout a wide range of initial glucose con centrations, but cell diameter and volume in-
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Fig, 2 , Cell dimensions in intact hyphal filaments of G, candidum in batch liquid culture at 30°. (a) Dimensions of apical cells during development of hyphal filaments , (b) Dimensions of non-apical cells. Cell number 1 is adjacent to the arthrospore, Each value is the mean of determination on 10 cells. Vertical lines represent 95 % confidence inter vals,
J. M.
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Fig. 4. Dimensions of successive non-apical cells formed prior to apical branch formation in G. candidum fragments in batch liquid culture at 30°. (a) Average dimensions of successive cells on mycelial fragments selected at random. (b) Sequence of cells produced by an individual apical branch (.) and by an individual lateral branch (0 ). Cell number 1 is adjacent (proximal) to an apical cell at the time of apical branch formation. Each value in Fig. 2a is the mean of determinations on 20 mycelial fragments. Vertical lines represent 95% confidence intervals. 6-2
Development of G. candidum
Fig. 5. Effect of temperature on cells of G. candidum grown on solid media. (a) Effect of temperature on cell diameter (., 25°; 0,30°) and cell volume (., apical cells, 2S'; 0, apical cells, 30°; _, subapical cells 2So; 0, subapical cells, 30°). (b) Effect of temperature on cell length (., apical cells; 0, subapical cells).Each value is the mean of determinations on 20 cells sea) and 120 cells S(b). Vertical lines represent 9S% confidence intervals where these exceed the height of the symbols.
creased with increasing glucose concentration. Similarly, the lengths of the subapical cells were constant throughout the wide range of initial glucose concentrations studied and cell diameter and volume increased with increasing glucose concentration. Cell dimensions were affected by a change in temperature, and the results in Fig. 5 illustrate this for cell diameter and volume at 25 and 30° and for cell length at 20, 25 and 30°. Cell diameter and volume were significantly less at 30° than at 25° for any given glucose concentration (Fig. 5 a). The length of apical and subapical cells decreased with increasing temperature (Fig. 5 b). The effects of temperature on non-apical cell dimensions and hyphal development in static liquid culture are shown in Table 2. Cell diameter and volume decreased with an increase in temperature from 20 to 30°; there was a significant decrease in cell length. At 20°, and an initial arthrospore concentration of 10 6 arthrospores ml- l , there was no significant difference between the mean number of septa formed at the time of
apical branch formation and the value obtained at 30°. However, at the lower temperature there was a marked increase in length of the non-apical cells, and as a result the mean filament length at the time of formation of the first apical branch was almost double that at 30°. DISCUSSION
When G. candidum is grown either in steady-state glucose-limited chemostat culture or on solid media with low initial glucose concentrations the cell dimensions and hyphal extension rates are similar. Pirt (1973) has plotted data for the reciprocal of the colony radial growth rate (K r ) of various fungi against the reciprocal of the growthlimiting substrate concentration in nutrient agar and obtained a linear relationship, except where K, was near the theoretical maximum. The results were taken to indicate that there was no substantial difference in the resistance to nutrient diffusion between hyphae and media in the peripheral
J. M. Smith and P. M. Robinson growth zone of a fungal colony on agar and the re sistance in an agitated liquid culture. The similarity of the nutrient concent ration gradients was attributed to the rapid advance of the leader hyphae on solid media and to the restriction of the hyphal density at low su bstrate conc ent rations on solid media (T rin ci, 1969 ; Rob inson & Smith, 1979 ). In the present work th e near-identical pattern of cell and hyphal development in chernostat culture and on solid media reinforces the idea that the two environments are sim ilar . A con sistent pattern of hyphal de velopment occur red in young batch liquid cultures of G. candidum, Each arthrospore produced a single hyph a which branched api cally when five or more septa had been delimited. The ap ical cells increased in diameter and volume during this phase of development and successive non-apical cells were wider and of greater volume. Fiddy & Trinci (1976) have described the growth of a single primary branch of G. candidum on a solid medium. The apical cell reached its maximum extens ion rate (E max) when five septa had been delimited. In the present work the progressive increa se in diameter and volume of each apical cell du ring dev elopment was also correlated with an increase in extension rate. There was a recurring cycle of development in olde r liquid cultures of G. candidum which contained numerous mycelial fragments. Each apical cell , whether produced as a lateral branch or as an apical branch, exhibited a progressive increase in diameter and volume until apical branch formation en sued. The cycle was then repeated by each new apical cell. There are some features of the effect of temperature on th e development of cells and hyphae of G. candidum which are cons tant , irrespe ctive of the culture conditions. In ch em ostat culture, for a given specific growth rate, both hyphal d iameter and cell volume increase as temperature decreases from 30 to 25° (Robinson & Sm ith , 1979). Apical and su bapical cells are short er at the lower temperature and more cells form lateral branches. \Vhen the temperature of a chernostat culture is lowered at a fixed dilution rate, the dilution rate becomes a larger fraction of th e attainable value of the maximum specific growth rate (I'ma x) and the organism approaches a physiological state
characteristic of unrestricted growth. The re sults ind icate th at the cell s and hyphae of G. candidum also approach a morphological state characteristic of unrestri cted growth when the temperature of a chemo stat culture is lowered. On solid med ia, for a given initial glucose concentration, hyphal diameter and cell volume decrease with an increase in temperature; cell length decreases. In batch liquid cultures hypha1 diameter, cell length and cell volume also decrease with an increase in temperature. There was a signi ficant effect on hyphal development when the organism was cultured at a lower temperature. Although the hyphae were full y developed at the time that eight septa had been delimited, both the time tak en for full dev elopment and the length of the hypha at full dev elopment were greater at 20° than at 30" due to the lower E max of the apical cell s and the increase in length of the non-apical cells.
REFERENCES
FIDDY, C. & TRtNCI , A. P. J. (1976). Nuclei, septation , branching and gro wth of Geotrichum candidum , Jou rnal of General Microbiology 97, 185-192. PIRT, S. J. (1973). Estimation of substrate affiniti es (K s values) of filamentous fung i fr om colony growth ra tes . J OllJ'llal of General M icrobiology 75, 245-247. R OBINSON, P . M . & SMITH, J. M . (1976). M orphogenesis and growth kinetics of G eotrichum candid um in continuous cult ure . Tran sactions of the British M y cological S ociety 66, 413-420 . ROBI NSON , P. M . & SMITH, J. M. (1979) . D evelopment of cells and hyphae of Geot richum candidum in chernos ta t and bat ch cultur e. T ransactions of the B riti sh M y cological Society 72, 39-47. STEELE, G . C. & TRI NCI , A. P . J. (1975). M orphology and growth kinetics of hyphae of differentiat ed and undifferentiated m ycelia of Neurospora crassa. J Ollrnal of G eneral M icrobiology 91, 362-368. TRINCI, A. P . J. (1969). A kinetic study of the gr owth of A spergillus nidulans and other fungi. [ourn al of General Microbiologv 57, 11-24. TRINCI , A. P . J. (197 1). Influence of the width of the peripheral growth zone on the radial growth rate of fungal coloni es on solid media. JOlll'llal of Gen eral Microbiology 67, 325-344. TRINCI , A. P. J. (1974). A study of the kinetics of hyphaI extension and branch initiation of fungal m ycelia . ] ourllal of General Microbiology 81, 225-236.
(R eceiv ed f or publication 19 February 1979 )
Printed in Great Britain
DEVELOPMENT OF SOMATIC HYPHAE OF GEOTRICHUM CA NDIDUM By
J. M . SMITH
A ND
P. M . ROBI NSO N
Department of B otany , The Queen's University, B elfast BT7 I N N , Northern Ir eland
H ypha e of Geotrichum cand idum Lk ex Pers. ha ve sim ilar ext ensio n rate s and cell dimension s when gro wn either in steady-s tate glu cose-limited ch ernostat cult ure or on solid med ia with low initial glu cose conce ntrations. In both method s of culture th e mycelium is undifferent iated and hyphal development is inco mplete. In liquid media wh ich permit full hyphal development there is a recurrent cycle of development in whi ch each apical cell branches d ichotom ou sly when five or more non ap ical cells have been pr oduced . During each cycle th ere is a grad ual increase in diameter and volume of the api cal cell and each successive non-ap ical cell produced is wider and of greater volume. The mycelium is undifferentiated. The effect of temperature on cell dimensions and hyphaI development is discussed for chemostat culture, batch liquid culture and culture on solid media. In most case s an increase in temperature is associat ed with a decrease in h yphal diameter and cell volume. In an earlier communi cation (Robinson & Smith, 1979) th e development of Geotrichum candid um Lk ex Pers. was described for glucose-limite d che most at culture and for cult ure on soli d med ia. In both cultur e con di tic ns the apica l an d non-apical cells increase d in diam eter and volume as th e speci fic growth rate of th e culture inc reased . These trends were integra ted within a d istinct pattern of hyph al development and, depending on th e con ditions of culture, mycelial differentiation . The present in vestigation involves a m ore detailed comparison of hyph al development of G. candidum in chemostat culture, on solid media and in batch liquid culture. The effect of temperature on hyph aI development is also consi dered . MAT ERIALS AN D M E TH O D S
The same strain of G. candidum used in earlier stud ies (Robin son & Smith, 1976, 1979) was maintained on slopes of Oxoid malt extract agar at 25 °C. For chemostat culture the art hrospores were washed from slopes into the following med ium [g(181) - 1] : D-glucose, 9'0 ; KH 2P04, 3'4; N a2 HP04. 12H20, 8'9; (N H 4)2 S04, 6'0; MgS04 . 7 H20 , 0'2 5; CaCI 2, 0'05 ; ZnS04 . 7H 20, 0'2 ; MnS04 • 7H 2 0 , 0'02 ; CuS0 4 • 5H 2 0, 0'005 ; F eSO•. 7H20, 0'1 ; Na2S04, 0'5 ; Na M 004.2H20 , 0 '005 ; disod ium ethy lenediami ne tetraacetic acid (EDT A), 0 ·6. The glu cose solutio n was ste rilized separately from th e rest of the medium. The chem ostat was a modular fermenter (A. E. G allenk amp & Co. Ltd, T echnico House, London , En gland ) which was modified for use with filamentou s fun gi and
which had a working volume of 0'5 I. The glu coselim ited cul ture was maintained at pH 6'0 ± 0' 1 by th e automatic addition of 0'1 M- NaOH. The culture was agi tated by an impeller and the temperature was controlled at 25 or 30 .L 0'5°. The air flow through the culture was ma intained at 0·6 I m in- I. Solid medi a were prepared by the additio n of Oxoid NO. 1 agar (10 g 1-1) to the same medium as used in th e chernos tat . The level of gluc ose was varied as required. For batch culture in liquid media, aliquots (25 ml ) of chemos tat med ium with a glucose con centration of 10 g I - I were steri lized in 500 ml conical flasks. The med ia were inoc ulated to give an art hrospo re concentra tio n of 10 6 art hros pores rnl" '. H yphal and cell dimensions were determ ined from enl arg ed pr int s of photogr aphs. Straight lines were fitt ed by regression analysis to data in Figs 2, 4 and 5· RESULTS
Correlation of cell dimensions in differ ent grow th conditions
In earlier experime nts (Robinson & Sm ith , 1979) neither mycelial differentiation nor complete hyphal development was detected in gluc oselim ited chemostat cultures of G. candidum under steady-state condi tio ns. Since steady-state cond ition s were establishe d over a relatively narrow range of low res idu al glu cose con centrations, an y parallels in cell dimensions , h yphaI development and growth kinetics m ight be expe cted between chemos tat cultu res and cult ures on solid me d ia
00°7-1536/80'2828-5670 $01.00 © 1980 T he British Mycological Society
Development of G. candidum
160
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Hyphal extension rate (Mill h-1 ) Fig. 1. Cell dimensionsand hyphal extension rates of G. candidum at 30° in chemostat culture and on solid media. Open symbols represent data from chemostat culture; closed symbols represent data from culture on solid media. Apical cells (e,O), non-apical cells (_,0). Each value is the mean of determinations on 20 cells. Vertical lines through symbols represent 95 % confidence intervals where these exceed the height of the symbols.
which contain low initial concentrations of glucose. The cell dimensions recorded for mycelial fragments from chemostat cultures and for colonies on solid media were almost identical for any particular hyphal extension rate which was common to each method of culture. For example (Fig. 1, Table 1), there was good agreement between the dimensions of cells from cultures grown at 30° at a specific growth rate of 0'25 h- I in the chemostat and at an initial glucose concentration (so) of 10 mg I-Ion solid media, and from cultures grown at a specific growth rate of 0'40 h- I in the chemostat and at an initial glucose concentration of 25 mg I-Ion solid media. Similar correlations occurred at 25° (Table 1) between the dimensions of cells from cultures grown at a specific growth rate of 0'30 h- I in the chemostat and at an initial glucose concentration of 10 mg I-I on solid media. For each comparison the hyphal diameters, apical cell volumes, and subapical cell lengths and volumes, were not significantly different. The number of comparisons was limited by steady-state conditions only being possible in the chemostat over a narrow range of residual glucose concentrations and by the difficulty of achieving very low concentrations of carbon source in solid media.
In each comparison in Table 1 the colony radial growth rate (K r ) , which was determined by direct measurement over a period of seven days on solid media, agrees closely with the mean hyphal extension rate (E) for the corresponding chemostat culture. E was determined from the equation E
=
G«,
where G = length of the hyphal growth unit and a = specific growth rate. This equation was established by Steele & Trinci (1975) for undifferentiated mycelia of Neurospora crassa growing exponentially on glucose-limited solid media. The mycelium of G. candidum is undifferentiated in steady-state chemostat culture and the length of the hyphal growth unit varies with specific growth rate (Robinson & Smith, 1979). The appropriate value of the hyphal growth unit for each specific growth rate was substituted in the above equation to give the mean hyphal extension rates for the chemostat cultures. For colonies on solid media the specific growth rate of the main hyphae was calculated from the equation K, = wa, where w = the width of the peripheral growth zone (Trinci, 1971). The width of the peripheral growth zone was assumed equal to the length of
J. M.
Smith and P. M. Robinson
the hyphal growth unit, which was calculated as 150 !Lm from data published by Trinci (1974) for undifferentiated mycelia of G. candidum. Each calculated value for the specific growth rate of the cultures on solid media was almost identical to the specific growth rate for the chemostat culture at which comparable cell dimensions were obtained. The results indicate a similarity in cell dimensions and hyphal extension rate when mycelia of G. candidum are grown in steady-state conditions in a chemostat or on solid media at very low initial glucose concentrations. Under both conditions there is no significant difference between the cell dimensions of main and lateral hyphae, and the cultures can be regarded as undifferentiated (Robinson & Smith, 1979). Development in batch liquid culture Development of somatic hyphae was studied for cultures of G. candidum during growth for 15 h at 30° in a static liquid medium which permitted full hyphal development. The arthrospore density after inoculation was 10 6 arthrospores ml- I • Each germinating arthrospore produced a single main hypha and the apical cell increased in diameter and volume as hyphallength increased (Fig. 2a). Successive non-apical cells were also wider and of greater volume (Fig. 2b) but no trends were
Table
1.
161
detected in the length of either apical or nonapical cells as hyphal length increased. Each apical cell had the capacity to branch dichotomously at the time that five or more (usually eight) septa had been delimited and the majority of hyphae had not fragmented at this stage. The average length of the hyphal filaments was determined at selected times prior to apical branch formation (Fig. 3) and the results indicated that the extension rate of the apical cells increased throughout this phase of development. In cultures incubated at 30° there was no difference in non-apical cell dimensions, in filament length at the time of formation of the first apical branch, and in the number of septa at this time, between hyphal filaments resulting from an initial arthrospore concentration of either 10 4 or 10 6 arthrospores ml- I (Table 2). Numerous mycelial fragments were present in cultures which had been incubated for 24 h. Each fragment had the potential to branch apically and there was a progressive increase in the diameter and volume of apical cells and of the sequence of non-apical cells formed prior to apical branch formation (Fig. 4a). There was no trend in cell length. Immediately after apical branch formation the first subapical cell formed by each new apical cell was narrow and of low volume. Each new apical cell, and the succession of non-apical cells
Cell dimensions and growth rates of hyphae of G. candidum in continuous culture and on solid media 30"C
25"'
30"
,
J~
Chemostat (a =o'30 hI)
\
Solid Medium Chemostat (So = 10 mg 1-1) (a=o'4 O h- I )
Solid medium (so = 10 mg I-I)
Chemostat (a =o'25 h- I)
Solid medium (So = 25 mg I-I)
3'7°::: 0'34
3'12±O'07
3'02±O'17
3'4 0±O'09
3'29±O'14
234± 18
190±12
209±8'3
191±9'5
208± 13
25 16±597
1452:::::: 1°9
1494± 164
1726-'::113
177 1±219
56'O±3'5
49'7::;::2'7.
5 1'2,1::2'7
44'2:::2'7
52'5±3'1
602±126
383±29
367±45
403±3 2
44 6:1:48
41'0
47'6t
47'0
67'6t
68'0
Hyphal width (/lm) 3,87=°'16* Apical cell length (/lm) 152::,,8'8 Apical cell volume (/lm 3) 1788_: 158 Subapical cell length (/lm) 53'OL 3'3 Subapical cell volume (/lm 3) 626 : 68
Meanhyphal extension rate (/lm h- I) Specific growth rate (h- I)
4 1'4t
0'3°
O'27~
0'25
0'31 ~
0'40
0'45~
*95% confidence intervals, t Calculated from E= Ga, where G = length of hyphal growth unit in chemostat (Robinson & Smith, 1979). ~ Calculated from Kr=wa, where w=G=150 /lm (Trinci, 1974), 6
MYC
74
Development of G. candidum
162 Table
2.
Hyphal development and average dimensions of non apical cells of G. candidum in static liquid culture
Spore density (arthrospores Temperature Cell length (aC) ml- 1) (pm) 20 2'78 x 10' 129'4 ± 13'9* 2'78 x 1O' 30 73'8 ± 7'3 2'78 x 104 66'o ± 6'3 3°
Cell diam (pm)
Cell volume (pm 3)
4'83 ±o'23 4'43 ±O '18 4'53 ±O'16
2371 ± 219 1149±1 27 1064± 88
Number of septa in filament 8'18 ±l '03 T82 ±O'99 9'12 ±1 '64
Length of filament (lim) 1230 ± 105 672 ± 52 611 ± 128
*
95% confidence intervals. Each result is the mean of determination s on 11 hyphal filaments, each of which had produced a single apical branch .
which were produced, showed the same increase in diameter and volume already described. This recurrent cycle of development was a constant feature of all hy phal fragments and could be identified in hyphae which formed eit her as lateral branches or as ap ical branches (F ig. 4b).
Effect of temperature on development of cells and hyphae In chemostat culture a shift in temperature from 25 to 30° results in several developmental changes
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"
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for a given specific growth rate. The diameter of apical and subapical cells decreases, as does the volume of the apical cells . Apical and subapical cells are longer, and fewer cells form lateral branches (Robin son & Smith, 1979). The dimensions of apical and subapical cells of G. candidum on solid media were also affected by temperature. For a given temperature, the lengths of the apical cells did not differ significantly throughout a wide range of initial glucose con centrations, but cell diameter and volume in-
,
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0
.
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E
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3
4
No, of septa in hypha
5
6
2
3
4
5
Cell no ,
Fig, 2 , Cell dimensions in intact hyphal filaments of G, candidum in batch liquid culture at 30°. (a) Dimensions of apical cells during development of hyphal filaments , (b) Dimensions of non-apical cells. Cell number 1 is adjacent to the arthrospore, Each value is the mean of determination on 10 cells. Vertical lines represent 95 % confidence inter vals,
J. M.
Smith and P. M. Robinson
600 500
400
E
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00 .:
300
~
co oJ::
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»
200
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\00
0
7
6
5
9
8
Incubation time (h) Fig. 3. Increase in hyphal length of germlings of G. candidum during batch liquid culture at 30°. Each value is the mean of determinations on 10 germlings. Vertical lines represent 95% confidence intervals.
(a)
'"E
(b)
•
2·0
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~i~~ 98765432 Cell no.
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98765432 Cell no.
Fig. 4. Dimensions of successive non-apical cells formed prior to apical branch formation in G. candidum fragments in batch liquid culture at 30°. (a) Average dimensions of successive cells on mycelial fragments selected at random. (b) Sequence of cells produced by an individual apical branch (.) and by an individual lateral branch (0 ). Cell number 1 is adjacent (proximal) to an apical cell at the time of apical branch formation. Each value in Fig. 2a is the mean of determinations on 20 mycelial fragments. Vertical lines represent 95% confidence intervals. 6-2
Development of G. candidum
Fig. 5. Effect of temperature on cells of G. candidum grown on solid media. (a) Effect of temperature on cell diameter (., 25°; 0,30°) and cell volume (., apical cells, 2S'; 0, apical cells, 30°; _, subapical cells 2So; 0, subapical cells, 30°). (b) Effect of temperature on cell length (., apical cells; 0, subapical cells).Each value is the mean of determinations on 20 cells sea) and 120 cells S(b). Vertical lines represent 9S% confidence intervals where these exceed the height of the symbols.
creased with increasing glucose concentration. Similarly, the lengths of the subapical cells were constant throughout the wide range of initial glucose concentrations studied and cell diameter and volume increased with increasing glucose concentration. Cell dimensions were affected by a change in temperature, and the results in Fig. 5 illustrate this for cell diameter and volume at 25 and 30° and for cell length at 20, 25 and 30°. Cell diameter and volume were significantly less at 30° than at 25° for any given glucose concentration (Fig. 5 a). The length of apical and subapical cells decreased with increasing temperature (Fig. 5 b). The effects of temperature on non-apical cell dimensions and hyphal development in static liquid culture are shown in Table 2. Cell diameter and volume decreased with an increase in temperature from 20 to 30°; there was a significant decrease in cell length. At 20°, and an initial arthrospore concentration of 10 6 arthrospores ml- l , there was no significant difference between the mean number of septa formed at the time of
apical branch formation and the value obtained at 30°. However, at the lower temperature there was a marked increase in length of the non-apical cells, and as a result the mean filament length at the time of formation of the first apical branch was almost double that at 30°. DISCUSSION
When G. candidum is grown either in steady-state glucose-limited chemostat culture or on solid media with low initial glucose concentrations the cell dimensions and hyphal extension rates are similar. Pirt (1973) has plotted data for the reciprocal of the colony radial growth rate (K r ) of various fungi against the reciprocal of the growthlimiting substrate concentration in nutrient agar and obtained a linear relationship, except where K, was near the theoretical maximum. The results were taken to indicate that there was no substantial difference in the resistance to nutrient diffusion between hyphae and media in the peripheral
J. M. Smith and P. M. Robinson growth zone of a fungal colony on agar and the re sistance in an agitated liquid culture. The similarity of the nutrient concent ration gradients was attributed to the rapid advance of the leader hyphae on solid media and to the restriction of the hyphal density at low su bstrate conc ent rations on solid media (T rin ci, 1969 ; Rob inson & Smith, 1979 ). In the present work th e near-identical pattern of cell and hyphal development in chernostat culture and on solid media reinforces the idea that the two environments are sim ilar . A con sistent pattern of hyphal de velopment occur red in young batch liquid cultures of G. candidum, Each arthrospore produced a single hyph a which branched api cally when five or more septa had been delimited. The ap ical cells increased in diameter and volume during this phase of development and successive non-apical cells were wider and of greater volume. Fiddy & Trinci (1976) have described the growth of a single primary branch of G. candidum on a solid medium. The apical cell reached its maximum extens ion rate (E max) when five septa had been delimited. In the present work the progressive increa se in diameter and volume of each apical cell du ring dev elopment was also correlated with an increase in extension rate. There was a recurring cycle of development in olde r liquid cultures of G. candidum which contained numerous mycelial fragments. Each apical cell , whether produced as a lateral branch or as an apical branch, exhibited a progressive increase in diameter and volume until apical branch formation en sued. The cycle was then repeated by each new apical cell. There are some features of the effect of temperature on th e development of cells and hyphae of G. candidum which are cons tant , irrespe ctive of the culture conditions. In ch em ostat culture, for a given specific growth rate, both hyphal d iameter and cell volume increase as temperature decreases from 30 to 25° (Robinson & Sm ith , 1979). Apical and su bapical cells are short er at the lower temperature and more cells form lateral branches. \Vhen the temperature of a chernostat culture is lowered at a fixed dilution rate, the dilution rate becomes a larger fraction of th e attainable value of the maximum specific growth rate (I'ma x) and the organism approaches a physiological state
characteristic of unrestricted growth. The re sults ind icate th at the cell s and hyphae of G. candidum also approach a morphological state characteristic of unrestri cted growth when the temperature of a chemo stat culture is lowered. On solid med ia, for a given initial glucose concentration, hyphal diameter and cell volume decrease with an increase in temperature; cell length decreases. In batch liquid cultures hypha1 diameter, cell length and cell volume also decrease with an increase in temperature. There was a signi ficant effect on hyphal development when the organism was cultured at a lower temperature. Although the hyphae were full y developed at the time that eight septa had been delimited, both the time tak en for full dev elopment and the length of the hypha at full dev elopment were greater at 20° than at 30" due to the lower E max of the apical cell s and the increase in length of the non-apical cells.
REFERENCES
FIDDY, C. & TRtNCI , A. P. J. (1976). Nuclei, septation , branching and gro wth of Geotrichum candidum , Jou rnal of General Microbiology 97, 185-192. PIRT, S. J. (1973). Estimation of substrate affiniti es (K s values) of filamentous fung i fr om colony growth ra tes . J OllJ'llal of General M icrobiology 75, 245-247. R OBINSON, P . M . & SMITH, J. M . (1976). M orphogenesis and growth kinetics of G eotrichum candid um in continuous cult ure . Tran sactions of the British M y cological S ociety 66, 413-420 . ROBI NSON , P. M . & SMITH, J. M. (1979) . D evelopment of cells and hyphae of Geot richum candidum in chernos ta t and bat ch cultur e. T ransactions of the B riti sh M y cological Society 72, 39-47. STEELE, G . C. & TRI NCI , A. P . J. (1975). M orphology and growth kinetics of hyphae of differentiat ed and undifferentiated m ycelia of Neurospora crassa. J Ollrnal of G eneral M icrobiology 91, 362-368. TRINCI, A. P . J. (1969). A kinetic study of the gr owth of A spergillus nidulans and other fungi. [ourn al of General Microbiologv 57, 11-24. TRINCI , A. P . J. (197 1). Influence of the width of the peripheral growth zone on the radial growth rate of fungal coloni es on solid media. JOlll'llal of Gen eral Microbiology 67, 325-344. TRINCI , A. P. J. (1974). A study of the kinetics of hyphaI extension and branch initiation of fungal m ycelia . ] ourllal of General Microbiology 81, 225-236.
(R eceiv ed f or publication 19 February 1979 )