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Branch production and fragmentation in the conidia of Pseudozyma prolifica Wiebe, Marilyn Gail 1986

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BRANCH PRODUCTION AND FRAGMENTATION IN THE CONIDIA OF PSEUDOZYMA PRQLD7ICA By MARILYN GAIL WIEBE B.Sc, The University o f Saskatchewan, 1984 A THESIS SUBMITTED IN PARTIAL FULiFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department o f Botany) We accept t h i s thesis as conforming -—teethe required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1986 © Marilyn G a i l Wiebe, 1986 \ I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f P> OT/IAJY The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 D a t e fa^J 12 tfn DE-6 (3/81) i i ABSTRACT The hyphomycete Pseudozyma p r o l i f i c a Bandoni was grown i n batch and continuous l i q u i d c u l tures t o determine the influence o f growth rate and n u t r i t i o n on conidium development. In batch culture, a period o f elongation and branch formation, followed by fragmentation, was t y p i c a l l y observed. The stage of branch formation was almost completely eliminated when the amino acids phenylalanine, glutamic acid, or asparagine were substituted f o r n i t r a t e . Substituting c i t r i c acid f o r glucose had a s i m i l a r e f f e c t . Branch formation was enhanced i n sucrose + n i t r a t e medium. In batch cultures, branched growth occurred a t the s t a r t o f the exponential growth phase. In continuous culture, the extent o f branching was dependent on the s p e c i f i c growth rate. Maximal branching was observed a t growth rates near the maximum. Growth by elongation and "bud" formation was predominant a t low growth rates, as a t the end o f the l o g phase i n batch cultures. The conidia were a l s o examined using fluorescence and el e c t r o n microscopy. Staining with wheat germ agglutinin conjugated t o Fluorescein Isothiocyanate indicated that some i n t e r c a l a r y growth occurs. i i i TABLE OF CONTENTS Abstract i i Table of Contents i i i L i s t o f Tables i v L i s t o f Figures v Acknowledgements v i i A. Introduction 1 B. Methods and Materials 4 1. Inoculum • . 4 2. Batch Growth 5 3. Fluorescence 9 4. Ele c t r o n Microscopy 9 5. Continuous c u l t u r e 10 6. Nutrient Studies 12 C. R e s u l t s — D e s c r i p t i v e 14 1. Light Microscopy 14 2. Fluorescence Microscopy . 14 3. E l e c t r o n Microscopy 17 D. Results—Growth Rate and Nutrition 24 1. Growth Rate . 24 2. N u t r i t i o n — B a t c h 30 3. Nutrition—Continuous c u l t u r e 37 4. N u t r i t i o n — C o n i d i a l Growth Unit 42 5. Temperature 45 E. Discussion—Descriptive 48 F. Discussion—Branching 54 G. Dis c u s s i o n — N u t r i t i o n 59 H. Summary 65 References 66 Appendix I 71 Appendix II 73 i v LIST OF TAELES Table I — Chi-square values comparing data from days 2-4(5) a t d i f f e r e n t d i l u t i o n rates and nutrient conditions 26 Table II — Distribution o f cells/conidium at D = 0.06/h, 0.15/h and 0.25/h. (% conidia with x c e l l s , where x = number o f c e l l s ) 29 Table U I — Chi-square values f o r combined branch frequencies o f duplicate runs a t D = O.l/h, 0.2/h and 0.29/h. a = 0.05 38 Table IV — Chi-square comparison o f a) sucrose and GN6 cultures and b) glycine and GN6 cultures. a = 0.01 38 Table V — Average conidia 1 growth units (p) a t D = 0.1/h, 0.2/h, and 0.29/h i n glucose + NO3 #6, sucrose, c i t r i c acid, glycine and glutamic acid media 46 Appendix I — Media and Solutions 71 Defined Media 71 Conn's Trace Elements 72 Malt-Yeast-Peptone 72 Phosphate Buffered Saline 72 V LIST OF FIGURES Figure 1 — Developmental sequence o f P. p r o l i f i c a . A. Blastoconidia inoculated i n t o f r e s h medium. B-D. Growth and branch formation. E. Onset of fragmentation. F. Arthroconidia, some producing single blastoconidia 2 Figure 2 — System o f aerated f l a s k s designed for batch experiments 6 Figure 3 — Arrangement o f f l a s k s on cooling p l a t e t o determine temperature e f f e c t s 7 Figure 4 — Chemostat designed f o r c u l t u r e o f P. p r o l i f i c a 11 Figure 5 — Fragmentation o f P. p r o l i f i c a conidia growing on MYP agar. Observations made at 1 hour i n t e r v a l s 15 Figure 6 — Changes i n the frequencies o f branched and unbranched conidia o f P. p r o l i f i c a during the f i r s t six days o f batch growth i n the chemostat column. 16 Figure 7 — P. p r o l i f i c a conidia labeled with WGA-FITC. Pairs show fluorescence and bright f i e l d illumination. Fluorescent micrographs show l a b e l l i n g o f conidium tips, branch i n i t i a l s , septa, secession scars, swollen c e l l s , and l o c a l i z e d fluorescence along the main conidium axis, (magnifcation: a l l p a i r s x 1890, except center l e f t x 1500. bottom l e f t x 1200.) 19 Figure 8 — U l t r a s t r u c t u r e of P. p r o l i f i c a showing t y p i c a l mitochondria, l i p i d bodies, vacuole, and septum. (x 17,000) 22 Figure 9 — Septum i n young blastoconidium o f P. p r o l i f i c a (x 19,000) 22 Figure 10 — Arthroconidium with two secession scars. (x 11,000) 22 Figure 11 — Septal pore with Vforonin bodies (x 24,500) 22 Figure 12 — Young conidium—vacuole shows a dense outer region and opaque inner region, (x 17,500) 22 Figure 13 — Mature conidium showing vacuole with granular contents, (x 12,800) 22 v i Figure 14 — Changes i n the branch frequencies of P. p r o l i f i c a conidia as the d i l u t i o n r a t e i s increased from D=0.06/h to D=0.28/h. Conidia were grown on GN6 medium 28 Figure 15 — Growth curves f o r P. p r o l i f i c a i n GN6 and fo r media i n which N, P, and Mg/s were reduced by h a l f . (Conidium concentration adjusted t o compensate f o r changes i n the extent o f branching, as described under methods.) 31 Figure 16 — The e f f e c t o f L-Sorbose on the morphology o f P. p r o l i f i c a conidia. Samples were taken a t 24 hour i n t e r v a l s 33 Figure 17 — P. p r o l i f i c a grown on glucose + N H 4 C I . A. A f t e r 1 day. B. A f t e r 5 days 35 Figure 18 — P. p r o l i f i c a conidium branch frequencies when grown on sucrose + NO3 medium 40 Figure 19 — P. p r o l i f i c a conidium branch frequencies when grown on c i t r i c acid + NO3 medium 41 Figure 21 — P. p r o l i f i c a conidium branch frequencies when grown on glucose + glycine medium 43 Figure 21 — P. p r o l i f i c a conidium branch frequencies when grown on glucose + glutamic acid medium 44 Appendix II — Batch Nutrient Studies 73 Figure 1 — Growth on GNI medium 74 Figure 2 — Growth on sucrose + N O 3 medium 75 Figure 3 — Growth on sorbose + NO3 medium 76 Figure 4 — Growth on c i t r i c acid + NO3 medium 77 Figure 5 — Growth on glucose + NH4 medium 78 Figure 6 — Growth on phenylalanine medium 79 Figure 7 — Growth on glucose + p r o l i n e medium 80 Figure 8 — Growth on glucose + glycine medium 81 Figure 9 — Growth on glucose + cystine medium 82 Figure 10 - Growth on glucose + asparagine medium 83 Figure 11 - Growth on glucose + glutamic acid medium . . 84 v i i ACKNCWEDGEMENTS I would l i k e t o express my appreciation t o my supervisor, Dr. R. J . Bandoni, f o r h i s encouragement and advice throughout the researching and writing o f t h i s thesis. I am a l s o g r a t e f u l t o several other people i n the department f o r t h e i r encouragement— p a r t i c u l a r l y t o L. Samuels and Dr. F. Molina f o r advice and assistance with the fluorescence and el e c t r o n microscopy. I thank Dr. P. S. S. Dawson f o r introducing me t o the concept of continuous c u l t u r e and giving me the incentive t o continue studying i n the f i e l d o f mycology. I am a l s o thankful f o r the scholarship awarded t o me by the Natural Sciences and Engineering Research Council o f Canada f o r these years o f study. - 1 -A. JJSITRODUCTION The hyphomycete Pseudozyma p r o l i f i c a Bandoni grows i n yeast-l i k e conidial colonies which may eventually generate hyphal growth. Both a r t h r i c and b l a s t i c conidia are produced ( f i g . 1), depending on the c u l t u r e conditions. A r t h r i c conidia a r i s e from the fragmentation o f blastoconidia which have elongated and become septate and branched. The arthroconidia do not generally form branches, but e i t h e r elongate or produce blastoconidia. These new blastoconidia can continue t o produce more blastoconidia, but do not develop branches unless t r a n s f e r r e d t o f r e s h media. The same pattern o f development was observed on a v a r i e t y o f media (Bandoni, 1985). This research was undertaken t o determine how growth rate and n u t r i t i o n a f f e c t the pattern and to f u r t h e r characterize c o n i d i a l development. In P. p r o l i f i c a , the balance between branch formation and fragmentation determines the developmental pattern observed. Thus the process of fragmentation i s important i n understanding conidial growth. A r t h r i c conidium production has been w e l l characterized i n the hyphomycete Geotrichum candidum. The conidia are formed by the septation o f hyphae a f t e r the cessation o f growth (Cole and Samson, 1979). Conidia are released when the septa s p l i t and the outer w a l l i s ruptured, leaving scars a t the conidia ends. Much of the recent research r e l a t i n g fungal d i f f e r e n t i a t i o n t o n u t r i t i o n and growth rate has been done with Geotrichum  candidum (Allermann e t a l . , 1983). G. candidum i s considered a u s e f u l organism f o r studying d i f f e r e n t i a t i o n i n submerged cul t u r e as the hyphae fragment f r e e l y and p e l l e t formation does not occur - 2 -A. Biastoconidia inoculated i n t o f r e s h medium. B-D. Growth and branch formation. E. Onset o f fragmentation. F. Arthroconidia, some producing s i n g l e blastocoriidia. - 3 -(Trinci and Collinge, 1974). P e l l e t formation introduces undesirable heterogeneity i n t o the cu l t u r e and may be a problem with other filamentous fungi ( P i r t and Callow, 1959). P. p r o l i f i c a i s a s i m i l a r l y suitable fungus f o r study i n l i q u i d culture, although the conidia may not be s t r i c t l y comparable t o hyphae. Nitrogen and carbon concentrations were found t o be important i n determining spore production and morphology i n Geotrichum  candidum (Park and Robinson, 1969; T r i n c i and Collinge, 1974; Kier e t a l . , 1976; Quinn e t a l . , 1981). The concentration and nature o f these nutrients i s known t o be important i n the d i f f e r e n t i a t i o n o f other fungi a l s o (Allermann e t a l . , 1983; Garraway and Evans, 1984) and i t was expected t o a f f e c t branch formation i n P. p r o l i f i c a . Since the substrate composition a f f e c t s s p e c i f i c growth r a t e (/Anderson e t a l . , 1975), continuous cu l t u r e was used t o separate the e f f e c t s o f n u t r i t i o n and growth rate. Sporulation i s enhanced a t low growth ra t e s i n G. candidum (Robinson and Smith, 1976) and i n other fungi (Righelato e t a l . , 1968; Ng e t a l . , 1973). I t has a l s o been noted t h a t mycelial fragments are more highly branched a t higher growth ra t e s (Katz e t a l . , 1972; Morrison and Righelato, 1974; Robinson and Smith, 1976). Geotrichum candidum d i f f e r s from P. p r o l i f i c a i n being a predominantly hyphal fungus. The arthroconidia are produced by hyphal fragmentation. As l i t t l e i s known about the conidial growth o f P. p r o l i f i c a , i t may not be s t r i c t l y comparable t o hyphal growth. Fluorescence and e l e c t r o n microscopy were u t i l i z e d t o gain some perspective on conidial growth. - 4 -B. METHODS AND MATERIALS A c u l t u r e o f Pseudozyma p r o l i f i c a (R. J . Bandoni #7293) was maintained on malt-yeast-peptone (MYP) agar p l a t e s (see appendix 1 f o r composition o f media). Light microscopy was done using a L e i t z phase contrast microscope. Observations o f conidial growth on agar were made d i r e c t l y using a p l a s t i c p e t r i dish with only a th i n layer o f agar. Conidia were spread over p a r t o f the agar and covered with a s t e r i l e c o v e r s l i p . A hole was cut i n the p e t r i dish cover f o r the microscope objective, providing semi-sterile conditions. The p l a t e was set up on a microscope with a drawing-tube and observations made over a number o f hours. 1. Inoculum Culture tubes (25 ml) containing 13 ml glucose + NO3 medium #5 (GN5—appendix 1) were inoculated d i r e c t l y with conidia from an agar p l a t e . A f t e r 24h, 1 ml o f t h i s l i q u i d culture would be tr a n s f e r r e d t o 13 ml f r e s h medium and allowed t o grow f o r another 24h or u n t i l the inoculum was needed. Flasks f o r aerated batch cultures were inoculated with 0.7-1.0 ml l i q u i d inoculum, depending on the c u l t u r e density. The inoculum f o r each f l a s k o f a given batch experiment was removed from the same cult u r e tube t o insure uniformity. Continuous c u l t u r e s were inoculated with 20 ml l i q u i d inoculum. Inoculum c u l t u r e s were taken through 2 t r a n s f e r s a t 24h i n t e r v a l s , as above. On the second transfer, 1 ml o f c u l t u r e would be t r a n s f e r r e d t o each of 2 f r e s h c u l t u r e tubes, t o provide the needed 20 ml f o r the following day. 24-29h cultures were - 5 -used t o incoluate the chemostat. P r i o r t o use, both tubes would be checked f o r contamination. To determine how consistent t h i s method o f inoculum preparation was, the o p t i c a l density o f the inoculum was measured using a Bausch & Lomb Spectronic 20. The absorbance a t wavelength = 360nm varied between 0.04 and 0.12. 2. Batch Growth Two systems were used t o f o l l o w the progression o f growth i n batch culture: a system o f aerated f l a s k s and an aerated column which could a l s o be used as a chemostat. The aerated f l a s k s were set up as shown i n Fig. 2. /Air was passed through a cotton f i l t e r and two 500 ml f l a s k s o f d i s t i l l e d H2O. The a i r then passed through the four c u l t u r e f l a s k s , providing both oxygen and mixing. Each cu l t u r e f l a s k would contain 150 ml medium. Samples were removed from a f l a s k with a s t e r i l e wire loop. The samples were then examined microscopically and the number o f branches on 100-200 individual conidia determined. Fewer conidia were examined i n c u l t u r e s of low density. This system was used t o determine the e f f e c t o f temperature on conidial branching. Each f l a s k was f i l l e d with 150 ml glucose + NO3 medium #6 (GN6) and inoculated with l i q u i d inoculum as prepared above. In the c o n t r o l experiment, a l l four f l a s k s were incubated a t room temperature and observations were made over a 4 day period. The experiment was then repeated, with the four f l a s k s being incubated on a copper cooling p l a t e a t temperatures of 11 C, 16 C, 21 C and 26 C (see Fig. 3). Observations were made over an 8 day period, during which time the temperatures on the surface o f the p l a t e remained constant within l e s s than h a l f a a i r pump Figure 2 — - System of aerated flasks designed for batch experiments. Figure 3 — Arrangement o f f l a s k s on cooling p l a t e t o determine temperature e f f e c t s . - 8 -degree. Temperatures within the f l a s k s were checked when the cu l t u r e s were terminated and found t o be a h a l f degree warmer than those on the surface of the pl a t e . A narrower temperature range was a l s o considered. Observations were made over a 6 day period f o r temperatures o f 19-20 C, 20-21 C, 21-22 C, and 22-23 C. The same system was a l s o used t o determine the l i m i t i n g nutrient i n GN6 medium. The f i r s t f l a s k contained the complete medium, whereas each subsequent f l a s k contained medium i n which the concentration o f one nutrient had been reduced by h a l f . Nitrogen, phosphorous, magnesium/sulfur and glucose were each considered i n t h i s manner. To determine the concentration o f conidia i n a c u l t u r e 0.05 ml, 0.025 ml, or 0.013 ml culture were removed from a given flask, spread on an agar-coated sl i d e , and the number o f conidia counted. The average number of branches was determined by counting the branches/conidium i n a separate sample taken a t the same time. The concentration o f l i v i n g matter was estimated by multiplying the average number of branches by the number o f conidia. The progression of growth i n batch cu l t u r e was al s o followed i n the chemostat f o r comparison with the continuous c u l t u r e studies. Medium (GN6) was prepared and 190 ml was drained into the c u l t u r e column. The column was inoculated with 20 ml (24h) inoculum and growth was followed over an 8 day period. The number of branches/conidium was determined f o r 200 conidia each day and 100 conidia were measured to estimate the conidial growth unit. - 9 -3. Fluorescence Fluorescence microscopy was used t o l o c a l i z e areas o f c h i t i n synthesis. Liquid c u l t u r e s were prepared using the same medium as f o r inoculum preparation. Approximately 6-8 ml o f cul t u r e were passed through a 3um m i l l i p o r e f i l t e r . The f i l t e r disk was rinsed with 5-10 ml d i s t i l l e d water and then incubated i n wheat germ agglutinin (WGA) conjugated t o Fluorescein Isothiocyanate (FTTC) (SIGMA Chemical Company) i n phosphate buffered saline (pH 7.4). A f t e r 1-2 h incubation, s l i d e s were prepared and examined with a L e i t z Dialux 20 EB fluorescent microscope using f i l t e r 12 (excitation range 450-490 nm, emitted wavelengths greater than 515 nm). 4. E l e c t r o n Microscopy For examination of conidial u l t r a s t r u c t u r e , conidia were grown i n l i q u i d c u l t u r e on MYP medium. One day-old conidia were c o l l e c t e d from 25 ml tubes containing 13 ml medium. Three day-o l d conidia were c o l l e c t e d from aerated f l a s k s containing 100 ml medium. The 2-wk o l d conidia were rinsed from the surface of MYP agar p l a t e s . The conidia were concentrated i n t o p e l l e t s by cent rifugation. The c e l l s were fixed i n 2% glutaraldehyde + 1% osmium tetroxide (0sO 4) f o r 2 h and post fixed i n 2% 0sO 4 f o r 1 1/2 h. NaCacodylate b u f f e r pH 7.4 (0.05 M, with 0.001 M C a C l 2 added) was used f o r washing the c e l l s between fixations. Dehydration was c a r r i e d out i n methanol, followed by anhydrous propylene oxide. PolyBed 812 was used t o embed the 3 day-old and 2-wk o l d conidia. Spurr's r e s i n was used f o r the 1 day-old conidia. Thin sections - 10 -were post-stained with uranyl acetate (40 min) and lead c i t r a t e (10 min). The sections were examined using a Zeis EM 9S-2 el e c t r o n microscope. 5. Continuous Culture A chromatography column was modified i n t o a chemostat t o c o n t r o l growth rate (Fig. 4). The medium reservoir had a capacity t o h o l d 11 l i t e r s of medium. Medium was drained into the column by gravity flow or by the use o f a MasterFlex p e r i s t a l t i c pump (Cole-Parmer Instrument Company). The C-Flex tubing used had an inside diameter o f 0.030 inches. A variable-speed drive was used with the pump, which allowed flow-rates t o be monitored between 0.2 - 1.5 ml/min. Medium entered the column near the bottom and was c i r c u l a t e d by a i r which entered through the sintered glass disk. A Si l e n t Giant aquarium pump was used t o pump a i r from the environment through a cotton f i l t e r and a f l a s k of d i s t i l l e d H 2 O i n t o the column. A 0.7mm lay e r o f gla s s beads (0.5 mm and 1.0 mm diameter) covered the sintered glass disk t o prevent extensive fungal growth i n the pores o f the disk. Samples could be removed from the middle o f the column, avoiding possible heterogenous conditions a t the base and a t the top. A three-way stopcock enabled the sample tube t o be emptied o f c u l t u r e a f t e r a sample had been taken, t o ensure that the next sample would be taken from the cult u r e i n the column. Harvest was removed from the surface o f the cult u r e . In the f i r s t column the l e v e l was set f o r 210 ml; i n the second column f o r 240-250 ml. - 11 -medium Input a i r f i l t e r harvest cu l t u r e vessel s i n t e r e d glass disk d i s t i l l e d water Figure 4 — Chemostat designed for culture of P. prolifica. - 12 -Prio r t o inoculating the column, 180-220 ml medium was drained from the medium rese r v o i r i n t o the column by gravity flow. The column was then inoculated using a 20 ml hypodermic syringe, injecting the conidia through the inoculation p ort a t the top o f the column. Aeration was st a r t e d and the cu l t u r e was allowed t o grow on batch f o r about 17 h. I t would then be set on t o continuous, the flow rate being c o n t r o l l e d by the p e r i s t a l t i c pump. Samples were removed d a i l y over a 4-5 day period. The approximate concentration of conidia i n the chemostat was determined by counting the number o f conidia i n 0.05 ml, 0.025 ml, or 0.013 ml spread over an agar-coated s l i d e . The number o f branches on each of 100-200 conidia was then determined. For the i n i t i a l growth r a t e studies, the number o f c e l l s per conidium was a l s o determined. C e l l counts were not made f o r the nutrient studies, but conidium length was measured f o r 50 conidia o f each sample t o provide an estimate o f the conidial growth unit. Growth r a t e experiments were conducted f o r d i l u t i o n rates o f 0.06/h, 0.08/h, 0.15/h, 0.20/h, 0.25/h, and 0.29/h. The d i l u t i o n rate was c a l c u l a t e d as flow rate/volume, which i s the f r a c t i o n o f the t o t a l c u l t u r e volume which changes i n 1 h. When the cu l t u r e i s i n a steady state, the s p e c i f i c growth rate o f the organism equals the d i l u t i o n r a t e (Herbert e t a l . , 1956). 6. Nutrient Studies The e f f e c t o f the nature o f the carbon or nitrogen source on coni d i a l branching was studied i n both batch and continuous c u l t u r e . For batch studies, the system o f aerated f l a s k s i l l u s t r a t e d i n Fig. 2 was used. GN6 medium was always placed i n - 13 -the f i r s t f lask, f o r reference. Each of the three following f l a s k s could then contain a d i f f e r e n t medium, and the r e s u l t s would be compared with the GN6 standard. The media (see appendix 1) were developed t o maintain the same carbon and nitrogen concentrations as i n the reference medium. This a l s o maintained the r a t i o o f C/N of 7.9:1. The only exception t o t h i s was i n glucose + NO3 medium #1(GN1), i n which the carbon concentration i s increased and the nitrogen decreased t o give a r a t i o o f 63.5:1. In media where carbon and nitrogen sources other than glucose and NO3 were used, only one nutrient was varied a t a time. Sucrose, c i t r i c acid and sorbose were used as carbon substitutes. Ammonium, glycine, glutamic acid, phenylalanine, asparagine, proline, and cystine were used as nitrogen substitutes. Growth i n each medium was followed over an 8 day period. Samples were taken d a i l y and the number of branches/conidium determined f o r 100-200 conidia. For continuous culture, four media were chosen t o be compared with GN6: sucrose + NO3, c i t r i c acid + NC^, glucose + glycine, and glucose + glutamic acid. Comparisons were made a t d i l u t i o n rates of 0.1/h, 0.2/h, and 0.29/h. Duplicate runs were done with GN6 a t each of these d i l u t i o n rates, as a c o n t r o l . - 14 -C. RESULTS—Descriptive 1. Light Microscopy P. p r o l i f i c a produces y e a s t - l i k e conidia 1 colonies when grown on agar. The conidia vary g r e a t l y i n siz e and shape, depending on the age o f the c u l t u r e and the p a r t o f the colony which i s examined. Simple conidia, consisting o f 1-4 c e l l s with no branches, are found i n a l l c u l t u r e s . Branched conidia are a l s o found, generally a t the growing edges o f the colony. Observations o f growth on agar show the development o f branches and the fragmentation o f branched conidia (Fig. 5). In l i q u i d cultures, the changes i n the extent o f branching can be followed by counting the number o f branches/conidium over a number o f days. When t h i s was done f o r the batch culture i n the chemostat, i t was found that the number o f branches/conidium increased between 24-48 h and then began t o decrease (see Fig. 6). By day 5, 25.5% o f the conidia were uribranched and by day 8, 49.5% were uribranched. The same pattern was observed when growth i n the aerated f l a s k s was observed, although the s p e c i f i c branch frequencies varied considerably, depending on the condition o f the inoculum (see appendix 2). 2. Fluorescence Microscopy Conidia o f P. p r o l i f i c a s t a i n w e l l with WGA-FITC. There was no autofluorescence i n the 450-490 nm excitation range. A f t e r 1-2 h incubation i n WGA-FITC, growing centers fluoresced b r i g h t l y . Longer incubation times r e s u l t e d i n an even more intense fluorescence at the growing s i t e s and some fluorescence of a l l c e l l w a l l s . Figure 5 — Fragmentation o f P. p r o l i f i c a conidia growing on MYP agar. Observations made at 1 hour i n t e r v a l s . - 16 -EXTENT OF BRANCHING ON DAYS 1-6 BATCH GROWTH ao-i I I D A Y 4 mm D A Y t» »—< DAY • 2 3 4 S S 7 B R A N C H E 5 / C O N I D I U M IO Figure 6 •— Changes in the frequencies of branched and unbranched conidia of P. pro l i f i c a during the f i r s t six days of batch growth in the chemostat column. Fluorescence was not restricted to branch tips, but was often seen at the base of branches and at septa, as well. Fig. 7 shows a number of conidia and the areas of fluorescence. Fluorescence i s seen in 3-4 main areas. Newly forming buds and young buds often fluoresce brightly over their entire surface. In slightly older buds, either the tip or the sides w i l l fluoresce brightly. Septa generally fluoresce and there is often a zone of fluorescence around them. Where a conidium has fragmented at a septum, the truncated ends of the two newly formed conidia generally fluoresce. Rings marking proliferative growth also fluoresce. Swollen c e l l s fluoresce—thus there is often fluorescence at the base of a newly formed branch or around a c e l l which has recently formed a branch. In some conidia, there w i l l also be fluorescence around the base where a branch has grown, even though neither the parent c e l l nor the branch has obviously swelled. Occasionally, localized fluorescence is also seen along the main axis of the conidium, even when there is no swelling. In conidia where some tips fluoresce, there are generally other tips which do not and in some conidia no tips show fluorescence. The walls of old or dead conidia fluoresce very brightly. 3. Electron Microscopy A simultaneous glutaraldehyde + O S O 4 fixation followed by a post-fixation in O S O 4 provided good preservation of membranous structures in 3 day-old conidia. The same fixation schedule did not preserve the mitochondrial, nuclear or vacuolar membranes in 1 - 18 -Figure 7 — P. p r o l i f i c a conidia labeled with WGA-FITC. Pairs show fluorescence and bright f i e l d i l lumination. Fluorescent micrographs show l a b e l l i n g o f conidium t i p , branch i n i t i a l s , septa, secession scars, swollen c e l l s , and l o c a l i z e d fluorescence along the main conidium axis, (magnification: a l l pa i r s x 1890, except center l e f t x 1500. bottom l e f t x 1200). c - 20 -day-old conidia, although the plasma membrane was w e l l preserved. Si m i l a r l y , the 2-wk o l d conidia were not adequately fixed using t h i s procedure and i n f i l t r a t i o n of the embedding material was poor. The conidia o f P. p r o l i f i c a have u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s s i m i l a r t o many filamentous hyphomycetes (Fig. 8). The young conidia are 1.5-2.5 um i n diameter, becoming more swollen as they mature (greater than 2.5 um diameter). The c e l l w a l l i s a l s o thinner i n younger conidia—being 60-100 nm thick i n 1 day-old conidia, but 100-150 nm i n more mature conidia, and exceeding 200 nm i n some conidia from the 2-wk o l d culture. The w a l l i s composed o f 2 or 3 laye r s . The innermost layer i s e l e c t r o n opaque and accounts f o r over 50% o f the w a l l thickness. The two outer layers are e l e c t r o n dense. The outermost l a y e r i s oft e n lacking along segments o f the w a l l , p a r t i c u l a r l y i n areas o f new growth. Blastoconidia from 1-day c u l t u r e s have th i n septa (42-90 nm) which often exhibit f o l d s or d i s t o r t i o n s (Fig. 9). This may r e f l e c t a f l e x i b i l i t y i n septa l e s s than 50 nm thick. The thicker septa are s t r a i g h t or only s l i g h t l y curved, suggesting that as the conidium grows i n diameter the septum thickens and becomes more r i g i d . Septa o f mature arthroconidia are 100-117 nm thick. The presence of a simple septal pore with Woronin bodies (Fig. 11) suggests a probable /Ascomycetous relationship. Secession scars are often present on the arthroconidia from 3-day cultures, but not on the blastoconidia o f 1 day-old cu l t u r e s . The conidia t y p i c a l l y p r o l i f e r a t e through the secession - 21 -Figure 8 — U l t r a s t r u c t u r e o f P. p r o l i f i c a showing t y p i c a l mitochondria, l i p i d bodies, vacuole, and septum, (x 17,000) Figure 9 — Septum i n young blastoconidium o f P. p r o l i f i c a . (x 19,000) Figure 10 — Arthroconidium with two secession scars, (x 11,000) Figure 11 — Septal pore with Woronin bodies, (x 24,500) Figure 12 — Young conidium—vacuole shows a dense outer region and opaque inner region, (x 17,500) Figure 13 — Mature conidium showing vacuole with granular contents, (x 12,800) - 22 -- 23 -scar and thus multiple scars can be present on one conidium (Fig. 10). Vacuoles appear t o be present i n a l l conidia, regardless of age. The nature o f the vacuolar contents, however, changes as the conidium develops. In young conidia there are two d i s t i n c t regions to the vacuole (Fig. 12)—an outer, somewhat dense region, and an inner, opaque area. There may a l s o be small opaque areas around the edge o f the vacuole. The opaque areas possibly represent l i p i d s or another storage product which can be mobilized t o provide energy t o the growing c e l l . This material i s not observed i n conidia from 3-day cu l t u r e s (Fig. 13). Instead, the vacuoles are f u l l o f a granular material. Ring-like structures were observed i n most vacuoles at t h i s stage. These are probably fingers o f cytoplasm extending i n t o the vacuole, suggesting a hi g h l y convoluted vacuolar shape. The granular material has l a r g e l y disappeared i n the aged conidia from 2-wk cultures. E l e c t r o n dense inclusions are t y p i c a l l y seen i n the vacuoles. These p a r t i c l e s are a l s o found i n the cytoplasm, but appear t o be sequestered i n the vacuoles. Glycogen and l i p i d are the two major storage compounds i n P. p r o l i f i c a . There i s an abundance of glycogen p a r t i c l e s i n the cytoplasm a t a l l stages o f growth. Di s t i n c t l i p i d bodies are present only i n 3 day-old arthroconidia. These are located near growing t i p s and septa. - 24 -D. RESULTS—Growth Rate & Nutrition 1. Growth Rate The chromatography column was an adequate culture v e s s e l f o r the l i q u i d c u l t u r e o f P. p r o l i f i c a . It's use as a chemostat was limited, however—as the conidia were able t o grow within the pores o f the sintered glass disk. This would e s t a b l i s h a source o f continual inoculum i n the c u l t u r e v e s s e l . Growth on the sintered disk was s i g n i f i c a n t l y reduced by placing a layer o f small glass beads over the disk. Using gl a s s beads, a c u l t u r e could be maintained i n the column f o r 7-8 days before the conidia became established on the disk. The glass beads did not i n t e r f e r e with the growth o f the conidia i n the medium. The beads did influence the flow o f a i r through the c u l t u r e medium. Larger a i r bubbles were produced i n the presence o f the beads than i n t h e i r absence. For dense cultures t h i s may have r e s u l t e d i n problems o f oxygen t r a n s f e r . P. p r o l i f i c a grows at low concentrations on synthetic media, so oxygen t r a n s f e r was not a problem i n the chemostat, even with the glass beads present. A f t e r inoculating the chemostat, the c u l t u r e would be l e f t on batch f o r approximately 17 h. There was always some c e l l death and considerable fragmentation o f the conidia i n response t o the change of environment from inoculation tube t o chemostat. The period o f batch growth enabled the c u l t u r e t o e s t a b l i s h i t s e l f i n the column. I t was considered that 4 x 10p conidia/ml was an adequate concentration o f conidia f o r s t a r t i n g continuous operation. - 25 -Two days o f growth at a constant flow rate were required before a steady state was established i n the culture column. The steady state could then be maintained f o r approximately 4 d a y s — u n t i l growth on the sintered disk began t o a f f e c t c u l t u r e conditions. Branches/conidium were thus counted each morning f o r days 2, 3 and 4 (and day 5 i f there was s u f f i c i e n t medium) o f continuous operation. The data c o l l e c t e d over these days was compared using a chi-square contingency t e s t t o show that the frequency o f conidia with 0-4 branches remained constant over t h i s time period. Only conidia with up t o 4 branches were considered f o r the t e s t , as at most flow rates the frequency o f conidia with more than 4 branches was r e l a t i v e l y low. I f the chi-square t e s t was sig n i f i c a n t , i t was assumed that a steady state had not been established i n the cu l t u r e column. With the exception o f runs which became contaminated and runs which were a f f e c t e d by severe temperature changes, i t was found that the data over days 2-4(5) was reproducible a t the a=0.05 or a=0.025 l e v e l o f significance (see Table I). Where there was no s i g n i f i c a n t difference i n branch frequencies the data from these days was pooled t o give a l a r g e r sample size. An estimate o f the conidium concentration i n the chemostat was a l s o determined d a i l y . Once a steady state i s attained i n a chemostat, the c e l l density should remain constant. The concentration o f conidia i n most cu l t u r e s was nearly constant during days 2-4. However, some cul t u r e s showed a general increase i n conidium concentration. This increase probably r e f l e c t s the growth of conidia on the sintered g l a s s disk and thus los s of a - 26 -Table I — Chi-square values comparing data from days 2-4(5) a t d i f f e r e n t d i l u t i o n rates and nutrient conditions, (a = 0.05) D 0.06/h 0.08/h 0.10/h 0.10/h 0.15/h 0.19/h X 2 c r i t i c a l X 2 15.73* 15.51 7.12 9.488 6.37 15.51 1.076 9.488 14.865 21.03 4.372 15.51 * acceptable a t a = 0.025 D 0.21/h 0.25/h 0.27/h 0.29/h 0.1/h s 0.2/h s X 2 c r i t i c a l X 2 15.22 21.03 17.78 21.03 8.847 21.03 11.756 15.51 2.675 15.51 6.06 21.03 s=sucrose D 0.3/h s 0.1/h c 0.2/h c 0.25/h c 0.1/h g 0.2/h g X 2 c r i t i c a l X 2 14.536 15.51 1.455 9.488 3.369 15.51 3.879 15.51 4.565 15.51 2.756 15.51 s=sucrose c=citrate g=glycine D 0.3/h g 0.1/h g l 0.2/h g l 0.3/h g l X 2 c r i t i c a l X 2 11.849 15.51 1.580 15.51 1.610 15.51 4.989 15.51 g=glycine gl=glutamic acid - 27 -steady state. Increases were most marked on days 4 and 5 and should not have a f f e c t e d the data on branch frequencies. As a control, the data c o l l e c t e d f o r batch growth i n the chemostat was analyzed s t a t i s t i c a l l y . For two separate runs the data f o r days 2-4 was shown t o be s t a t i s t i c a l l y very d i f f e r e n t (X 2 = 41.34 and X 2 = 133.36 where X28(a=o.05) = 15.51). This demonstrated that the constant pattern o f branching observed during continuous operation was the r e s u l t of c o n t r o l l i n g the growth rate, not the design of the cu l t u r e v e s s e l . The chemostat was run a t d i l u t i o n rates (D) o f 0.06/h, 0.08/h, 0.10/h, 0.15/h, 0.20/h, 0.25/h, and 0.29/h. From the graphs presented i n Fig. 14, i t i s apparent that there i s an increase i n the average number o f branches/conidium as the d i l u t i o n rate i s increased. At d i l u t i o n rates of 0.06/h, 0.08/h and 0.10/h, there i s a high frequency o f unbranched conidia. At D=0.15/h and D=0.20/h, the frequency o f unbranched conidia has dropped, and the frequency of conidia with 1 branch has increased. At D=0.25/h and D=0.29/h, there i s another s h i f t , increasing the frequency o f conidia with 2 and 3 branches. Although i t i s not as apparent from the graph, the data a l s o showed a general increase i n the frequency at which conidia with more than 5 branches were seen as D was increased. A s i m i l a r r e lationship was observed between the number of c e l l s per conidium and the growth rate. C e l l s were counted at d i l u t i o n rates of 0.06/h, 0.15/h and 0.25/h. At D=0.06/h there i s a high frequency o f s i n g l e - c e l l e d conidia. This corresponds t o the high frequency o f unbranched conidia. At D=0.15/h, 4- c e l l e d conidia are more frequent and a t D=0.25/h conidia of 5 or more - 28 -THE EFFECT OF DILUTION RATE ON BRANCH FORMATION Figure 14 — Changes in the branch frequencies of P. pro l i f i c a conidia as the dilution rate ia increased from D=0.06/h to D=0.28/h. Conidia were grown on GN6 medium. - 29 -Table TJ — Distxibution of cells/cxxiidium at D = 0.06/h, 0.15/h and 0.25/h. (% conidia with x cells, where x=number of cells). D = 0.06/h 0.15/h 0.25/h # cells/conidium  1 39.6 6.8 1.4 2 20.2 10.5 5.0 3 14.3 13.4 6.6 4 10.8 25.7 13.3 5 4.9 12.8 12.6 6 2.2 7.9 12.7 7 3.3 4.2 9.7 8 2.4 2.9 9.8 9 0.9 3.4 6.9 - 30 -c e l l s predominate (Table U). The increase i n c e l l number with increased branching indicates that septa are l a i d down r e g u l a r l y as the conidium grows and forms branches. 2. N u t r i t i o n — b a t c h GN6 medium was used as a c o n t r o l i n studying the a f f e c t o f the carbon and nitrogen source on co n i d i a l branching. This medium was used i n the i n i t i a l batch studies and the growth rate experiments so i t was considered an appropriate standard t o compare other media with. By reducing the various nutrients i n the GN6 medium by h a l f , and observing the e f f e c t s on c e l l concentration, i t was determined th a t magnesium or s u l f a t e was the l i m i t i n g nutrient i n the medium (see Fig. 15). Thus i n substituting other carbon or nitrogen sources f o r glucose or NO3, nutrients i n excess are being changed and growth i s s t i l l l i m ited by the Mg/S. Nitrogen becomes l i m i t i n g when i t s concentration i s reduced by h a l f (see Fig. 15) and t h i s would a f f e c t the growth on GN1 medium, where the C:N r a t i o has been increased by increasing the glucose and decreasing the nitrogen. Growth on GN1 medium (low nitrogen) showed reduced branch formation when compared with GN6 medium (Fig. 1, appendix 2). A f t e r 3 days of growth, 53.4% of the conidia were unbranched. I t was not u n t i l day 7 that the conidia growing on GN6 (high nitrogen) showed such a high percentage of unbranched conidia. Nor was there a t any time a high frequency o f conidia with 4 or more branches on low N medium, as seen during days 1 and 2 on the high N medium. The same trend was observed i n the nutrient l i m i t a t i o n - 31 -GROWTH OF Pseudozyma prolifica ON GN6 TO DETERMINE LIMITING NUTRIENT 450-1 T I M E d a y s Figure 15 — Growth curves for P. prolifica in GN6 and for media in which N, P, and Mg/S were reduced by half. (Conidium concentration adjusted to compensate for changes in the extent of branching, as described under methods.) - 32 -experiment when the nitrogen concentration was reduced by h a l f (C:N = 16:1). The extent o f branching was lower on the medium with the higher C:N r a t i o . Growth on sucrose + NO3 medium was s i m i l a r t o growth on GN6, but branching was s l i g h t l y increased (Fig. 2, appendix 2). This was most noticeable on day 2, when 31% of the conidia growing on sucrose had more than 10 branches, whereas only 7% o f those growing on glucose had more than 10. The differences were l e s s s t r i k i n g on days 3-8, but the extent o f branching diiriinished more slowly i n the sucrose cultures than i n the glucose cultures. Sorbose a f f e c t e d the general morphology o f the conidia more than the extent o f branching. There was, however, a longer l a g phase i n terms o f both c e l l concentration and branch formation. The c u l t u r e density remained low f o r the f i r s t 3-4 days. Maximal branching occurred on days 3 and 4, rather than days 1 and 2 as i n glucose. The data from day 3 on sorbose i s very similar t o day 2 on glucose. Subsequent changes i n the branching pattern p a r a l l e l those o f normal growth. On account o f the extended lag phase, branching appears t o be greater on sorbose than on glucose on days 3-8 (Fig. 3, appenidix 2). Fig. 16 shows diagrams o f how conidium morphology was a f f e c t e d by growth on sorbose—the conidia became progressively more contorted, developing i r r e g u l a r swellings. A dark pigment develops i n the c e l l s by day 9 i f the cultures are allowed t o continue growing. C i t r i c acid a f f e c t e d both branching and general conidium morphology. The branched conidia i n the inoculum seemed t o fragment and these arthroconidia then grew i n length rather than Figure 16 — The e f f e c t o f L-Sorbose on the morphology o f P. p r o l i f i c a conidia. Samples were taken at 24 hour i n t e r v a l s . - 34 -forming new branches. Thus the frequency o f branched conidia decreased r a p i d l y i n the f i r s t 3 days—and by day 3, 80% o f the conidia were unbranched (Fig. 4, appendix 2). These elongate conidia were narrower i n diameter than conidia grown on glucose medium. Ammonium was substituted f o r n i t r a t e i n the form of NH4CI. The medium was not buffered t o compensate f o r pH changes as the ammonium was metabolized. During the f i r s t 3 days growth was . r e l a t i v e l y normal, although the conidia had fewer branches than n i t r a t e grown conidia (Fig. 5, appendix 2). By day 3, the conidia were more branched than the c o n t r o l conidia, but they were a l s o s t a r t i n g t o show signs o f swelling. By day 5, the swellings were pronounced and the conidia were deformed i n appearance (Fig. 17). It was d i f f i c u l t t o determine the number of branches on these deformed conidia and no further data was c o l l e c t e d . When amino acids were substituted f o r n i t r a t e there was always a decrease i n the extent o f branching. In most cases the conidia appeared t o fragment i n response to being transferred to the new media. Eventually a few branches would develop, followed again by fragmentation. The extent o f i n i t i a l fragmentation and subsequent development o f branches varied greatly, depending on the amino acid used (see Figs. 6-11, appendix 2). In glycine, glutamic acid, asparagine and phenylalanine media the i n i t i a l fragmentation res u l t e d i n over 40% of the conidia being unbranched on day 1. There were l e s s than 5% unbranched conidia on day 1 i n GN6 cultures. Fragmentation was l e s s complete i n cystine and p r o l i n e media. In cystine, only 20% o f the conidia - 35 -Figure 17 — P. p r o l i f i c a grown on glucose + NH4CI. A. A f t e r 1 day. B. A f t e r 5 days. - 36 -were unbranched and 50% had 1 branch. In proline, only 10% were unbranched, while 28% o f the c u l t u r e s t i l l had 1 branch and 32% had 2 branches. The per cent o f unbranched conidia i n the phenylalanine medium continued t o increase over the course o f the 8 days with no evidence o f new branches being formed (Fig. 6, appendix 2). On proline, a l s o the extent o f branching decreased p r o g r e s s i v e l y — although i n i t i a l fragmentation had not been as pronounced. The frequency a t which unbranched and single-branched conidia were observed increased more slowly than i n GN6 medium. Thus by day 8 the p r o l i n e c u l t u r e showed a lower percentage o f unbranched conidia than the n i t r a t e c u l t u r e (Fig. 7, appendix 2). However, the conidia did not look as healthy as those grown on n i t r a t e . Pigment began t o be produced on day 5. On other media, branch formation followed a l a g period. On glycine and cystine, the l a g period was r e l a t i v e l y short and maximal branch production was observed on day 2. Maximal branch production was, however, considerably reduced f o r both amino acids. Branches appeared t o develop over days 2 and 3 i n the glycine c u l t u r e . There was a predominance o f single-, 2-, and 3-branched conidia on these days, which was followed by fragmentation (Fig. 8, appendix 2). The percentage o f unbranched conidia increased t o 89% by day 7. Brown pigment was observed i n the culture on day 5. In cystine, fragmentation was already apparent on day 3, and the frequency o f unbranched conidia had increased further on day 4 - 37 -(Fig. 9, appendix 2). However, day 5 showed a drop i n the per cent o f unbranched conidia and an increase i n the per cent o f conidia with 2 branches. Day 6 showed a high frequency (44.2%) o f conidia with 1 branch and day 7 a high frequency (31%) with 2 branches. Thus the pattern o f changes which occurred on days 1-4 appear t o be repeated on days 5-8. The cu l t u r e developed a pale yellow pigment. In both glutamic acid and asparagine, the l a g period was considerably longer. By day 2, fragmentation i n the asparagine c u l t u r e had r e s u l t e d i n over 80% o f the conidia being unbranched (Fig. 10, appendix 2) and only 11% having even 1 branch. By day 3, the per cent o f single-branched conidia had increased t o 25.5, suggesting new branch formation. Branch formation occurred over days 3 and 4 — t h e n fragmentation again re s u l t e d i n a predominance o f unbranched conidia. I t was not u n t i l day 5 that there was evidence o f new branch formation i n glutamic acid c u l t u r e s . The frequency o f unbranched conidia increased d a i l y during days 1-4, but then dropped on days 5 and 6 (Fig. 11, appendix 2). On day 7, the per cent o f unbranched conidia had increased once more. Dark pigment was formed as the cu l t u r e aged. 3. Nutrition—continuous c u l t u r e The branch counts over days 2-4(5) for duplicate runs i n GN6 medium were s t a t i s t i c a l l y the same a t D=0.l/h and D=0.3/h (Table III). The var i a t i o n observed between the two runs at D=0.2/h i s probably explained by the f a c t that the flow rate was set s l i g h t l y slower f o r one run than the other, and the variation i n the data - 38 -Table m — Chi-square values for combined branch frequencies of duplicate runs at D = O.l/h, 0.2/h and 0.29/h. a = 0.05. D 0.1/h 0.2/h 0.29/h 0.2/h* X 2 9.175 99.312 22.561 11.67 c r i t i c a l X 2 26.296 36.415 36.415 13.28** * based on average branch frequencies, not individual totals ** a = 0.01 Table TV — Chi-square comparison of a) sucrose and GN6 cultures and b) glycine and GN6 cultures, a = 0.01. a) glucose—GN6 b) glycine—GN6 D 0.02/h 0.10/h 0.20/h 0.29/h X 2 20.277 13.30 9.369 12.413 c r i t i c a l X 2 13.28 13.28 13.28 13.28 from run 1 was greater than that from run 2. When the average percentages o f conidia with 0, 1, 2, 3, and 4 branches were compared using a chi-square t e s t they were found t o be s t a t i s t i c a l l y the same a t the a=0.01 l e v e l of significance. Thus the two runs were accepted as the same and i t was concluded that the branching patterns produced a t any one growth rate would be reproducible i f a l l other f a c t o r s remained constant. This a l s o showed that although the physiological condition o f the inoculum i s important i n producing consistent r e s u l t s , the procedure followed i n growing i t was adequate. Duplicate runs were not c a r r i e d out f o r the other media used, but good r e p r o d u c i b i l i t y was found over days 2-4 within each run (see Table I). In c i t r i c acid medium, however, a steady state was gener a l l y not attained u n t i l day 3 — s o only data from days 3-4(5) were used. Conidia grown on sucrose (Fig. 18) were generally more branched than those grown on glucose (Fig. 14). At D=0.l/h there was a high percentage o f conidia with 1 and 2 branches, but only 13% were unbranched. On glucose medium almost 30% of the conidia were unbranched. At D=0.2/h the majority o f conidia had one branch i n both sucrose and glucose cultures. The chi-square t e s t indicated that there i s a s i g n i f i c a n t difference i n the branch frequencies (Table IV). The differences were greater at D=0.29/h. Grown on sucrose, most conidia have 2 or 3 branches; on glucose, 1 or 2. Washout occurred at D=0.36/h. Branching was s i g n i f i c a n t l y reduced when c i t r i c acid was substituted f o r glucose (Fig. 19). There was a high percentage - 40 -BRANCH FORMATION IN SUCROSE MEDIUM AT D=0.10/h, 0.20/h and 0.29/h 30-i • i i . mm i i i p n i , . p *-i • i -mm »_L mm m i mm * L. mm i i _, mm 0 1 2 3 4 5 6 7 8 9 10 BRANCHES/CONIDIUM Figure 18 — P. pr o l i f i c a conidium branch frequencies When grown on sucrose + NO3 medium. - 41 -BRANCH FORMATION IN CITRIC ACID MEDIUM AT D=0.10/h, 0.20/h and 0.25/h 70-i 60-50-< Q Z o o 40-30-20-10-J I i Legend O D=0.10/h • 1 D=0.20/h \m D=0.29/h 7^1 ,pcn 0 1 2 3 4 5 6 7 8 9 BRANCHES/CONIDIUM — r -10 Figure 19 — P. pro l i f i c a conidium branch frequencies when grown on c i t r i c acid + NO3 medium. - 42 -of unbranched conidia a t a l l growth r a t e s — 5 5 % at D=0.l/h and D=0.2/h, 30% at D=0.25/h. There was e s s e n t i a l l y no difference i n the extent o f branching a t D=0.l/h and a t D=0.2/h. At D=0.25/h the frequency o f conidia with 3 or more branches increased. However, D=0.25/h was near the washout p o i n t — t h e concentration o f conidia i n the c u l t u r e dropped ten f o l d (to about 1 x 10^) i n the f i r s t two days o f continuous operation Branch formation on glycine medium was very s i m i l a r t o that on GN6 (Fig. 20). There were s l i g h t l y higher percentages o f conidia with low numbers o f branches, but the differences were not si g n i f i c a n t (Table IV). Thus a t D=0.l/h unbranched conidia were predominant, a t D=0.2/h single-branched conidia were, and a t D=0.3/h conidia with 2 branches. The response t o glutamic acid resembled that t o c i t r i c acid a t D=0.l/h (X 2 = 0.453 i s i n s i g n i f i c a n t a t a=0.05). The extent o f branching was reduced a t D=0.2/h i n comparison t o n i t r a t e medium, but was s l i g h t l y increased i n comparison with the c i t r i c acid c u l t u r e . There was s t i l l a predominance o f unbranched conidia, but the frequency had dropped from 55% t o 40%. There was an increase i n unbranched conidia a t D=0.29/h. Growth was sim i l a r t o th a t at D=0.l/h, with 57% unbranched conidia (Fig. 21). 4. N u t r i t i o n — c o n i d i a l growth unit The conidial growth unit was c a l c u l a t e d as the length o f a conidium divided by the number o f t i p s the conidium had. Thus unbranched conidia have 2 t i p s and single-branched conidia 3, etcetera. The average conidial growth unit was calculated f o r 50 conidia per sample. This average could a l s o be broken into the - 43 -BRANCH FORMATION IN GLYCINE MEDIUM AT D=0.10/h, 0.20/h and 0.29/h 45-1 40-35-30-< 25-O O O ^ 20-1 15-10-5-I fl Legend a D=0.10/h •i D=0.20/h V& D=0.29/h 2 3 4 5 6 7 8 BRANCHES/CONIDIUM Figure 20 — P. pr o l i f i c a conidium branch frequencies when grown on glucose + glycine medium. - 44 -BRANCH FORMATION IN GLUTAMIC ACID MEDIUM AT D=0.10/h, 0.20/h and 0.29/h 70 -i 60-50-< Q O O 40-30-20-10-L e g e n d CD D=0.10/h •i D=0.20/h Jm D=0.29/h -^t™—r-—i—T 2 3 4 5 6 7 8 BRANCHES/CONIDIUM — r 9 10 Figure 21 — P. pro l i f i c a conidium branch frequencies when grown on glucose + glutamic acid medium. - 45 -average growth unit f o r conidia with any given number o f branches. The co n i d i a l growth unit showed considerable v a r i a b i l i t y over days 2-4 (Table V) even though the branching pattern was constant during t h i s time. I t was generally lowest a f t e r 17 h o f batch growth, then increased when continuous operation was started. Growth rate does not appear t o a f f e c t the conidial growth unit g r e a t l y . N u t r i t i o n had a much more marked a f f e c t . The average co n i d i a l growth unit was i n the range 15.0-18.0u f o r glucose, sucrose and glutamate c u l t u r e s . In glycine, i t was lowered t o 12.5-16u. In c i t r i c acid i t was increased t o 18.0-24.0p. 5. Temperature P. p r o l i f i c a grows more slowly a t "low" temperatures than at "high". When the 4 cultures grown a t 11, 16, 20 and 25 C were compared, i t was found that the cu l t u r e density was lowest at 11 C and highest a t 20 C. The c u l t u r e growing a t 25 C was not as dense as the cu l t u r e a t 20 C, and the conidia developed i r r e g u l a r swellings by the 3rd day o f growth. When the d a i l y branch patterns o f these cultures were compared, they were not the same. The cu l t u r e grown at 11 C underwent more gradual changes than the other cultures. Maximal branching occurred on days 3 and 6, rather than on day 2. On day 7 the branch pattern was most s i m i l a r t o that observed on day 4 i n the 20 C cu l t u r e . At 16 C, maximal branching was observed on day 4. Subsequent changes i n branch frequency were comparable t o the changes which occurred on days 3-6 at 20 C. Growth at 20 C resembled growth at room temperature. Maximal branching was observed on days 1 and 2 - 46 -Table V — Average cxxiidial growth units (u) at D = O.l/h, 0.2/h, and 0.29/h in glucose + NO3 #6, sucrose, citric acid, glycine and glutamic acid media. glucose D 0.10/h 0.19/h 0.21/h 0.27/h 0.29/h batch day 1 day 2 day 3 day 4 13.72 14.42 16.57 15.08 16.10 13.91 15.58 16.43 14.92 15.82 12.90 16.68 15.90 16.69 16.28 16.24 16.75 17.81 16.27 16.69 13.28 16.59 17.42 17.97 16.68 sucrose citric acid D 0.10/h 0.20/h 0.29/h 0.10/h 0.20/h L 0.25/h batch day 1 day 2 day 3 day 4 14.34 15.13 16.61 17.44 17.18 16.24 15.98 17.34 16.07 17.22 12.36 16.52 17.14 16.65 15.40 14.47 20.82 17.96 21.47 20.70 16.45 18.16 23.82 21.00 21.95 14.76 20.62 21.59 24.13 21.90 glycine glutamic acid D 0.10/h 0.20/h 0.29/h 0.10/h 0.20/h 1 0.29/h batch day 1 day 2 day 3 day 4 12.74 14.57 12.54 14.98 13.48 15.13 13.57 13.70 14.55 16.14 12.36 16.20 13.96 14.18 16.15 16.80 15.95 15.53 15.02 16.20 16.07 16.18 17.71 15.99 18.97 18.16 21.74 27.75 20.93 23.47 - 47 -and decreased s t e a d i l y a f t e r that. Growth at 25 C was not normal. The conidia became swollen and the pattern o f branching showed l i t t l e change a f t e r day 3. There appears t o be an optimal temperature f o r growth that l i e s near 20 C. Room temperature generally fluctuated between 21 and 23 C. Cultures were grown a t 19-20, 20-21, 21-22, and 22-23 C t o observe difference i n t h i s temperature range. Maximal branching occurred on day 2 i n a l l 4 cultures. Although there were minor differences between the cultures, the branch patterns were e s s e n t i a l l y the same over days 3-6 (X 2 values are acceptable at a=0.01). This shows that the normal fluctuations i n room temperature w i l l have had l i t t l e e f f e c t on the r e s u l t s obtained. - 48 -E. DISCUSSION—De sc r i p t i v e Bandoni (1985) described the y e a s t - l i k e conidia 1 colonies of Pseudozyma p r o l i f i c a as "producing blastoconidia and/or fragmenting." The pattern whereby blastoconidia elongate, develop branches and fragment t o produce arthroconidia, which i n turn produce blastoconidia was then described. This pattern has now been given a quantitative aspect by determining changes i n the frequency o f unbranched and branched conidia over time. The conidia have been further characterized using fluorescence and e l e c t r o n microscopy. Branch counts from batch c u l t u r e s showed that a t any given time there w i l l be both branched and unbranched conidia present. I t i s the r e l a t i v e frequency o f conidia i n each category that changes as the cult u r e matures. Such a situation i s not unexpected and may simply r e f l e c t the heterogenous nature of a batch c u l t u r e and the v a r i a b i l i t y c h a r a c t e r i s t i c of the fungi. The development of a given conidium i s dependent upon both i t s environment and i t s ancestory (Powell e t a l . , 1967). As the environment i s continually being modified i n a batch culture, c e l l s a t d i f f e r e n t stages o f the c e l l c y c l e w i l l be subjected t o d i f f e r i n g environments upon completion of c e l l duplication. Such differences w i l l i n turn a f f e c t the subsequent growth o f the new e e l Is/conidia. That observations made from batch c u l t u r e represent a cross-section of conidia with s i m i l a r yet variable p h y s i o l o g i c a l states should be kept i n mind when considering the descriptions given here. - 49 -The branch counts showed an i n i t i a l l y high frequency o f multi-branched conidia (2-4 branches). As the culture matured, the frequency o f conidia with fewer branches would gradually increase, u n t i l most o f the conidia i n the c u l t u r e were unbranched. This can be explained i f the branched conidia fragment. Fragmentation was not observed d i r e c t l y i n l i q u i d culture, but was observed on agar. The presence of secession scars around the truncated ends o f unbranched conidia a l s o indicates that they were formed by fragmentation. In studies using WGA-FITC, the secession scars fluoresced, making them r e a d i l y v i s i b l e . In young cultures few conidia with scars were observed. In older cultures, secession scars were present on the majority o f conidia. This corresponds w e l l with a pattern where elongation and branch formation i s followed by fragmentation. Developing branches, septa, truncated ends formed by fragmentation, and swollen c e l l s a l s o fluoresce b r i g h t l y when stained with WGA-FITC. WGA binds s p e c i f i c a l l y t o N-acetylglucosamine and has been shown t o be s p e c i f i c f o r c h i t i n i n many fungi (Mirelman e t a l . , 1975; Barkai-Golan et a l . , 1978; Mendgen et a l . , 1985). Mirelman e t a l . (1975) demonstrated that WGA binds t o areas o f hyphal growth i n the Deuteromycete Trichoderma v i r i d e by comparing the hyphae stained with WGA-FITC and c u l t u r e s l a b e l l e d with % - a c e t a t e . % - a c e t a t e i s used t o mark growing zones i n t h i s fungus (Galun, 1972). Both methods l a b e l only hyphal t i p s and septa. Mirelman et a l . (1975) suggested that c h i t i n was most accessible i n the t i p s and septa, but became concealed by glucans i n the more mature hyphal walls. Further - 50 -study using soybean agglutinin and peanut agglutinin supported this hypothesis (Barkai-Golan et al., 1978). These lectins are specific for D-galactose or N-acetyl-I>galactosamine. They bind along mature hyphal walls, but not at the tips or septa, suggesting a layer of galactose residues (or galactans) over these surfaces. Such an arrangement is in agreement with current concepts about the structure of hyphal c e l l walls (Burnett, 1979; Wessels and Sietsma, 1981). Based on the results of Mirelman et a l . (1975) and Barkai-Golan et a l . (1978) some conclusions can be drawn about growth in P. prolifica. The staining of branch i n i t i a l s and the tips of young branches indicates apical growth i s occurring in these areas. This was anticipated, as hyphal growth i s restricted to the tip region (Marchant and Smith, 1968; Gooday, 1971; Galun, 1972). Fluorescence, however, i s not restricted to branch tips, nor do a l l tips show fluorescence in P. prolifica. The absence of fluorescence at some tips shows that not a l l branches present on a conidium are elongating. The duration of growth of any one branch may be under genetic control and may be dependent on the number of branches already present or the number of new branches forming. Fluorescence in regions other than the tips may be indicative of intercalary growth or that chitin i s more exposed in some parts of the conidium wall than others. The staining of secession scars i s an example of the latter. When two c e l l s of a conidium separate, the outer wall breaks and the inner wall layer is exposed. As chitin occurs primarily in the inner wall (Cabib, - 51 -1981), t h i s would make i t l o c a l l y more accessible t o the s t a i n . S i m i l a r l y , septa appear t o have a high l e v e l o f cl-iitin (Galun, 1972). However, not a l l septa fluoresce. As i t i s often the younger septa which fluoresce, one might assume that a l a y e r o f glucans or galactans masks the c h i t i n i n older septa. E l e c t r o n microscopy showed that the septa thicken as the conidia become more mature. Individual layers are not discernible, but the outer edges o f the septum are generally more e l e c t r o n dense than the middle. A s i m i l a r pattern has been observed i n other Ascomycetes and Basidiomycetes (Beckett e t a l . , 1974; Wessels and Sietsma, 1981). In the Basidiomycete Schizophyllum cummune the septum i s composed o f a chitinous s e p t a l p l a t e covered on both sides with a glucan-chitin complex (Wessels and Sietsma, 1979). I f t h i s i s a general pattern, the c h i t i n must become s u f f i c i e n t l y masked i n o l d e r septa t o account f o r the lack o f WGA binding. When a conidium fragments, the outer w a l l breaks and the septum s p l i t s . The end w a l l s o f the two adjacent c e l l s are each composed o f a half-septum derived from the inner w a l l layer. The e l e c t r o n micrographs show that a new outer w a l l i s eventually deposited. Before t h i s process i s complete, the chitinous m i c r o f i b r i l s of the inner layer would be more r e a d i l y stained with WGA-FITC than other parts o f the c e l l w a l l . This could account f o r the observed fluorescence o f truncated end c e l l s , these may a l s o be areas o f growth, i.e. as the conidium p r o l i f e r a t e s through the secession scar. Staining i s a l s o seen a t the base o f some branches, i n the zone around some septa, and i n patches along the main axis o f the conidium. As the inner w a l l i s not exposed i n these areas, the fluorescence may mark regions o f i n t e r c a l a r y growth. Observations o f growth on agar a l s o suggested th a t i n t e r c a l a r y growth occurred during the branching phase o f conidia 1 growth. The growth o f vegetative hyphae appears t o be e x c l u s i v e l y a p i c a l (Burnett, 1976; Gooday, 1983) although subapical growth can be i n i t i a t e d by shock treatments (Kate and Rosenberger, 1971). I t must be concluded t h a t growth and development i n P. p r o l i f i c a conidia i s not e n t i r e l y comparable t o hyphal growth. The fluorescence o f swollen c e l l s i s more l i k e l y an indication o f l a t e r a l growth and new w a l l deposition rather than o f i n t e r c a l a r y growth. Ruperez and L e a l (1986) found that the c h i t i n content i n Penicillium e r y t h r o m e l l i s w a l l s was highest i n very young and i n o l d cultures. As i t i s the mature conidia o f P. p r o l i f i c a which become swollen, fluorescence i n these regions i s i n agreement with the r e s u l t s o f Ruperez and L e a l . E l e c t r o n microscopy revealed that the c e l l w a l l thickens as conidia age. This thickening must thus involve c h i t i n synthesis as w e l l as the deposition o f mannans, glucans and galactans. Exandning the u 1 t r as t r u c t u r e o f blastoccriidia and arthroconidia a l s o revealed other physiological changes occurring as the conidia mature. Differences i n the vacuoles and storage products were most obvious. Park and Robinson (1970) reported changes i n the mitochondria and vacuoles between the arthrospore and somatic phases o f growth i n Geotrichum can did um. Mature arthrospores were characterized by short, o v a l mitochondria and prominent vacuoles. Growing c e l l s had filamentous mitochondria and smaller, more numerous vacuoles located near the growing tip. The association of numerous, small mitochondria with c e l l s of low metabolic activity was also noted by Garraway and Evans (1984). Such differences were not obvious in P. prolifica. However, swollen conidia generally did have smaller mitochondria than other c e l l s . As in G. candidum, vacuoles in P. prolifica appeared most prominent in older arthroconidia when examined by light microscopy. Electron microscopy revealed prominent vacuoles in conidia at a l l stages of growth examined and differences in the vacuolar contents seem to be more important. These differences probably represent changes in cel l u l a r metabolism. The catabolism of different storage products at different stages of development may significantly affect the range of growth rates within which a conidium can grow. In turn, the growth rate probably affects the type of storage product which is formed. - 54 -F. DISCUSSION—Branching That conidia produce branches when tra n s f e r r e d t o f r e s h medium, but fragment as the c u l t u r e matures and then do not appear capable o f again becoming multi-branched suggests that the growth o f branches i s dependent upon the growth rate and the nutri e n t supply. Transfer t o f r e s h medium allows the conidia t o grow a t a f a s t e r rate. The p h y s i o l o g i c a l changes which occur as the conidium once again becomes metab o l i c a l l y active somehow r e s u l t i n a switch from growth occurring as elongation, swelling and bud formation t o elongation and branch formation. A high growth rate appears necessary f o r t h i s change t o occur. However, while s t i l l i n the exponential stage of growth, fragmentation begins. Thus, growth rate i s not the sole f a c t o r determining branch formation. Fragmentation may be triggered by the build-up o f s t a l i n g products or may be g e n e t i c a l l y c o n t r o l l e d . To study the function of the growth rate i n c o n t r o l l i n g the extent o f branching, a chemostat was designed which was suited f or the growth o f P. p r o l i f i c a . A r e l a t i v e l y large inoculum was used t o reduce the length o f time the c u l t u r e required t o es t a b l i s h i t s e l f . As Veldkamp (1976) pointed out, the size of inoculum i s t h e o r e t i c a l l y o f no importance i n a continuous culture, but may be i n p r a c t i c e . Once a steady state i s established i n a chemostat, each generation develops i n the same environment as the previous generation (Powell e t a l . , 1967). The input from the inoculum becomes smaller with each successive generation. In the e a r l y generations, however, the inoculum might be expected to have some e f f e c t . Therefore i t i s necessary t o co n s i s t e n t l y inoculate with - 55 -conidia a t the same growth stage. The conclusions drawn from the data w i l l be applicable only f o r the inoculum used, although more extensive experiments should show that the same trends apply i n a l l situations. Use o f the chemostat was r e s t r i c t e d to r e l a t i v e l y short time periods. Two days were required a t most flow ra t e s f o r a steady st a t e t o be established, leaving 3-4 days i n which t o c o l l e c t data. Solomons (1972) a r b i t r a r i l y suggests a minimum operating time o f 1000 hours f o r a chemostat t o be c l a s s i f i e d as such. The c u l t u r e column used was not suited f o r extended continuous operation. Nevertheless a steady state could be maintained f o r a number o f days, which was considered s u f f i c i e n t f o r the purposes o f t h i s research. The data should be interpreted within the limitations o f the equipment. I t was necessary t o re-inoculate the column f o r each flow rate, but i t was shown that the r e s u l t s a t a given flow rate were reproducible. I t was was concluded that the individual runs were a l s o comparable. Based on data from chemostat cultures, i t appears that branch formation i s l i m i t e d by the growth rate. I t has been shown that there are fewer branched conidia at low growth rates than a t high. /AS i n batch growth, a range o f branched and unbranched conidia were observed a t a l l growth rates. There are two probable reasons why t h i s v a r i a b i l i t y e x i s t s . F i r s t , complete mixing was not attained i n the chemostat, so the c u l t u r e was not completely homogeneous. Medium was introduced a t the base o f the column and c i r c u l a t e d upwards with the a i r - 56 -b u b b l e s — a p a r t i a l gradient may have been established. The presence o f conidia on the sintered disk a f t e r several days has a s i m i l a r e f f e c t o f increasing the heterogeneity. As a r e s u l t , the d i l u t i o n r a t e i s not uniform throughout the column, enabling some organisms t o remain i n the column longer than others (Herbert e t a l . , 1956). The age range o f conidia i n the cu l t u r e i s increased i n an incompletely mixed system. Even under i d e a l conditions there i s a range o f c e l l ages i n a continuous c u l t u r e system (James, 1961). The system i s more uniform than a batch culture, but even i n a synchronized cu l t u r e there are c e l l s of d i f f e r i n g age (Dawson, 1980). In addition, i t i s obvious that i f a t D=0.3/h most conidia have 2 branches, there must a l s o be conidia with no or only one branch, which w i l l develop a second branch t o replace those conidia which are removed i n the harvest. Thus the chemostat can be used t o demonstrate tha t growth rate i s important i n determining branch formation, but the p i c t u r e i s blurred. Secondly, the genetic input o f the conidia must be considered. I t has been observed that fragmentation begins while a batch c u l t u r e i s s t i l l i n the exponential stage. S t a l i n g products do not have the oportunity t o b u i l d up i n a continuous culture, suggesting t h a t fragmentation i s c o n t r o l l e d by the c e l l s themselves. The c o r r e l a t i o n between growth rate and branch production i n P. p r o l i f i c a i s probably best understood i n terms o f l i m i t a t i o n . At slow growth rates (less than D=0.l/h) conidia are incapable of becoming multi-branched. Instead they form single branches which sece d e — a s i n l a t e r stages o f batch growth. Some conidia surpass - 57 -the average growth rate and are able t o r e t a i n the f i r s t branch and form more. As the growth rate i s increased, the l i m i t i n branch number a l s o increases. At D=0.15-0.2/h 1-2 branches can be sustained and a t D=0.3/h 2-4 branches can be. Fragmentation occurs i n a l l c u l t u r e s accounting f o r conidia with fewer branches. Some conidia remain i n the chemostat long enough t o produce seve r a l more branches accounting f o r the multi-branched conidia. Genetic v a r i a t i o n between conidia would a l s o a f f e c t the maximum number of branches a conidium could develop under given conditions, as would conidium h i s t o r y . A s i m i l a r relationship between growth rate and branch formation has been observed i n Geotrichum candidum (Robinson and Smith, 1976), Penicillium chrysogenum (Morrison and Righelato, 1974) and A s p e r g i l l u s niger (Katz et a l . , 1972). As hyphae grow by a p i c a l extension a t a l i n e a r rate yet increase exponentially, i t i s apparent that the number of branches must be increasing exponentially ( P i r t and Callow, 1960; Katz e t a l . , 1972). Further, the frequency of branching must be r e l a t e d t o the maximum growth r a t e and elongation r a t e of the fungus. As a r e s u l t of t h i s r e l a t i o n s h i p there appears t o be an average length of hypha associated with each t i p — t h e "hyphal growth unit" (Caldwell and T r i n c i , 1973; B u l l and T r i n c i , 1977). The hyphal growth unit was found t o be constant during the exponential phase o f growth, but the value varied at d i f f e r e n t growth rates and f o r d i f f e r e n t species ( B u l l and T r i n c i , 1977). This confirmed f o r a wide variety o f fungi that branch frequency i s dependent upon growth rate. - 58 -In Pseudozyma p r o l i f i c a the conidial growth unit does not vary with growth r a t e and thus d i f f e r s from the hyphal growth unit. This suggests that the extension rate i s r e l a t e d t o the s p e c i f i c growth r a t e i n a l i n e a r manner (Trinci, 1984). I t should a l s o be noted, that unlike hyphal fungi, exponential growth i n P. p r o l i f i c a does not necessitate an increase i n t i p number. Fragmentation produces conidia with fewer branches which continue t o reproduce by blastoconidium production. Fragmentation occurs on s o l i d medium as w e l l as i n l i q u i d culture. Hyphal fungi normally grow i n i n t a c t colonies—fragmentation r e s u l t i n g from s t r e s s i n submerged c u l t u r e . Fragmentation, however, can a l s o be r e l a t e d t o s p e c i f i c growth rate i n Geotrichum candidum (Robinson and Smith, 1976; Quinn e t a l . , 1981). G. candidum produces h o l o t h a l l i c conidia i n a manner sim i l a r t o P. p r o l i f i c a . More arthrospores were formed a t low d i l u t i o n r a t e s than a t high, although some were produced even a t high d i l u t i o n r a t e s . The occurrence of spores a t high growth rates and throughout batch c u l t u r e indicates that f a c t o r s other than s p e c i f i c growth rate contribute t o the regulation o f spore production (Quinn e t a l . , 1981). Quinn e t a l . (1981) suggest that sporulation i s continuous i n G. candidum and that low s p e c i f i c growth rates a c t t o enhance the process. In P. p r o l i f i c a enhanced fragmentation would contribute t o the i n a b i l i t y o f conidia t o become highly branched a t low d i l u t i o n rates. - 59 -G. DISCUSSION—Nutrition The e f f e c t s o f various carbon and nitrogen sources on growth and sporulation have been studied i n a number o f fungi (see Allermann e t a l . , 1983; Garraway and Evans, 1984). Substrate composition a f f e c t s the maximum s p e c i f i c growth rate o f an organism (Anderson e t a l . , 1975). When both n u t r i t i o n and growth rate have been considered, an int e r a c t i o n between t h e i r e f f e c t s has generally been observed (Smith and Anderson, 1973; Broderick and Greenshields, 1981; Quinn e t a l . 1981). The nature o f both the carbon and nitrogen source influenced the extent o f co n i d i a l branch formation i n P. p r o l i f i c a . I t was noted that growth rate and pigment production were a l s o a f f e c t e d . These e f f e c t s were studied i n Mg/S-limited cultures, with the exception o f the low nitrogen medium which was N-limited. The C:N r a t i o i n a l l other media was maintained a t 7.9:1. To maintain t h i s r a t i o i n amino-acid media, the glucose concentration had t o be reduced, so there i s a compounded e f f e c t o f substituting both the carbon and nitrogen source. Growth on low nitrogen medium was characterized by reduced branch formation or increased fragmentation. The implication i s that a high C:N r a t i o , low N concentration or nitrogen l i m i t a t i o n favour fragmentation. A low C:N r a t i o and available nitrogen would favour branch production. Nitrogen starvation has a l s o been shown t o increase fragmentation i n Geotrichum candidum (Park and Robinson, 1969; T r i n c i and Collinge, 1974) and may induce sporulation i n submerged cultures o f Asp e r g i l l u s niger i f there i s s u f f i c i e n t carbohydrate a v a i l a b l e (Broderick and Greenshields, - 60 -1982). Of the other media used, only i n sucrose + NO3 were conidia more highly branched than i n GN6. The differences observed i n batch c u l t u r e may have r e f l e c t e d differences i n the time required t o a t t a i n the maximum sp e c i f i c growth rate. However, Anderson e t a l . (1975) noted that most fungi can assimilate sucrose and grow a t rates comparable t o those on glucose. The extent o f conidial branching was a l s o s i g n i f i c a n t l y increased i n the chemostat cultures, snowing that the increase was independent o f growth rate. When grown a t D=0.2/h on sucrose the conidia had fewer branches than they had when grown a t D=0.l/h. This was not anticipated, as the batch cultures had shown the same pattern of development that was observed i n GN6 cultures. At D=0.29/h, the extent o f branching had increased t o the expected l e v e l . I t i s u n l i k e l y that the conidia go through two c y c l e s o f branch formation and fragmentation on sucrose. The low l e v e l o f branching a t D=0.2/h i s more l i k e l y the r e s u l t o f human er r o r i n medium preparation or an uncontrolled environmental factor. L-sorbose was observed t o i n h i b i t colony expansion i n Neuropsora and Syncephalastrum and i s frequently used t o reduce fungal growth (Tatum e t a l . , 1949). T r i n c i and Collinge (1973) observed growth o f Neurospora crassa on s o l i d and l i q u i d media to which sorbose had been added. On s o l i d media, the hyphal extension rate i s reduced, but the growth rate i s not affected. A corresponding increase i n branching r e s u l t s . In l i q u i d culture, there was no increase i n branch formation. The presence of - 61 -sorbose i n the medium did however increase the duration o f the l a g phase. Growth and branch formation was s i m i l a r l y delayed i n P. p r o l i f i c a . Development then p a r a l l e l e d that i n glucose culture, with no increase i n branch frequency. The change i n conidium morphology indicated that some form o f growth inhib i t i o n was occurring. Similar morphological changes were observed under other inhibitory conditions including low pH, high temperature and growth on cyanide. T r i n c i and Collinge (1973) suggested that L-sorbose may a c t by upsetting the balance o f synthesis and cleavage o f w a l l polymers, r e s u l t i n g i n w a l l softening. L o c a l softening o f the c e l l w a l l could lead t o the contorted growth and swollen branches observed i n P. p r o l i f i c a . Similar swellings were observed i n cultures o f Diplodia natalensis i n which c e l l w a l l synthesis had been inhibited with polyoxin D (Galun e t a l . , 1981). In contrast t o growth on glucose, sucrose, or sorbose, growth on c i t r i c acid inhibited branch formation, favouring growth by elongation and fragmentation. The maximum s p e c i f i c growth rate was lowered s i g n i f i c a n t l y — f r o m 0.36/h (in sucrose culture) t o 0.25/h. The increased conidial growth unit r e f l e c t s the lower extent o f branching. C i t r a t e i s one o f the sugars o f the t r i c a r b o x y l i c acid (TCA) c y c l e and in h i b i t s sugar metabolism by feedback in h i b i t i o n (Bohinski, 1979). In Aspergillus niger the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways are p a r t i a l l y repressed under c i t r a t e - l i m i t a t i o n while the TCA c y c l e i s not (Ng e t a l . , 1974). The p a r t i a l repression o f cata b o l i c pathways may account f o r the decreased s p e c i f i c growth - 62 -r a t e and increased fragmentation observed. I t i s frequently observed that nitrogen-limitation a f f e c t s sporulation (Allermann e t a l . , 1983; Garraway and Evans, 1984). The form i n which the nitrogen i s supplied i s a l s o important. N i t r a t e and ammonia are commonly used as inorganic N-sources; amino acids and proteins serve as organic sources. Apparent differences i n nitrogen metabolism may, however, be masking pH e f f e c t s (Shepherd and Carels, 1983). The swelling e f f e c t s observed i n NH4CI cultures o f P. p r o l i f i c a were probably caused by pH changes i n the medium. Conidia remained healthy and branched during the f i r s t days o f batch growth. I t was assumed that ammonium did not stimulate fragmentation and t h a t P. p r o l i f i c a was able t o use ammonium as sole nitrogen source within a l i m i t e d pH range. Ammonium was found to i n h i b i t fragmentation i n Geotrichum candidum—although the e f f e c t was compounded by pH e f f e c t s (Quinn e t a l . , 1981). Ammonium a l s o i n h i b i t s sporulation i n A s p e r g i l l u s niger (Smith and Anderson, 1973), Monascus spp. (Carels and Shepherd, 1977) and Bipolar i s maydis (Shepherd and Carels, 1983). The use o f an organic nitrogen source had a marked a f f e c t on conidium fragmentation i n P. p r o l i f i c a . Growth occurred on a l l amino acids tested, but the extent o f branching was s i g n i f i c a n t l y reduced on a l l but cystine and glycine. I t might be assumed that the unique p a t t e r n o f branch formation observed i n cystine c u l t u r e s r e s u l t e d from the increased S concentration. In a study o f nitrogen n u t r i t i o n o f B i p o l a r i s maydis race T, Evans and Black (1981) summarized growth and sporulation: "In general, amino acids with acidic side chains were the best sources f o r both growth and sporulation; those with uncharged polar side chains were moderately good, and those with nonpolar side chains l a r g e r than a methyl group were the poorest." Asparagine and glycine have been observed t o support good sporulation o f Helminthosporium spp. (Tarr and Kafi, 1968) and asparagine a l s o allows sporulation o f Geotrichum candidum (Quinn et a l . , 1981). I f fragmentation i s considered p a r a l l e l t o the sporulation i n these fungi, i t might be expected t o be highest i n glutamic acid, asparagine and p r o l i n e media, moderate i n glycine and ni t r a t e , and lowest i n ammonium and phenylalanine media. Other responses t o amino acids are, however, observed i n other fungi and are p a r t i a l l y dependent on the carbon source present (Garraway and Evans, 1984). In P. p r o l i f i c a , fragmentation was greatest on phenylalanine. This p o s s i b l y indicates an i n a b i l i t y t o metabolize the phenyl ring and hence severe carbon li m i t a t i o n . I t may a l s o r e f l e c t the production o f excess ammonia as the carbon i s metabolized f o r growth (Pateman and Kinghorn, 1976). Asparagine and glutamate both favoured fragmentation and supported only limited branch formation, whereas glycine was comparable t o n i t r a t e . The i n i t i a l fragmentation observed i n a l l amino acid cultures may r e f l e c t a delay i n the uptake of amino acids from the media. The i n i t i a l stages o f growth would thus be nitrogen-limited. Subsequent development would be dependent on the nature of the p a r t i c u l a r amino acid being metabolized and the s p e c i f i c growth r a t e attained by the fungus. An i n i t i a l lag i n nitrogen uptake could account f o r the discrepancy between batch and continuous - 64 -cultures o f P. p r o l i f i c a i n glycine. In batch culture, branching i s reduced i n comparison with the n i t r a t e culture. In continuous c u l t u r e there i s no difference. The decreases i n conidial growth unit indicates there are other metabolic differences involved. I t has been suggested by B u l l and T r i n c i (1977) and Shepherd and Carels (1983) that d i f f e r e n t i a t i o n i s a t l e a s t p a r t i a l l y regulated by the balance between the EMP, PP and TCA pathways and tha t t h i s balance i s a f f e c t e d by the nature o f the nutrient source. Nitrogen l i m i t a t i o n i n h i b i t s the PP pathway and t h i s s h i f t may favour sporulation rather than growth (Carels and Shepherd, 1977; Shepherd and Carels, 1983). Thus fragmentation would occur rather than branch formation i n amino acid and low-nitrogen media. C i t r a t e a l s o i n h i b i t s the PP pathway (Ng et a l . , 1974) and was observed t o favour fragmentation. The PP pathway i s enhanced a t higher growth r a t e s ( B u l l and Bushell, 1976), allowing branches t o form. /Although observations o f P. p r o l i f i c a support Shepherd and Carels' hypothesis, there i s evidence o f the PP pathway being stimulated during sporulation o f other fungi (B u l l and T r i n c i , 1977). - 65 -H. SUMMARY Growth o f P. p r o l i f i c a conidia i s a balance between branch formation and fragmentation, a f f e c t e d by growth rate and nutrient supply. Blastoconidia grow by a p i c a l extension and branch formation during the i n i t i a l stages o f batch growth. Limited i n t e r c a l a r y growth i s observed near septa and at the base o f some branches. Branch formation i s possible a t high growth rates on e a s i l y metabolized carbohydrates such as sucrose and glucose where nitrogen i s r e a d i l y a v a i l a b l e . Fragmentation occurs a t low growth rates, on media with low nitrogen a v a i l a b i l i t y , and on c i t r i c a c i d media. I t i s suggested th a t the repression o f the pentose phosphate pathway may enhance fragmentation. Arthroconidia are produced when two c e l l s o f a conidium separate a t the septum. The outer w a l l i s broken, but the inner w a l l remains i n t a c t . The arthroconidia either p r o l i f e r a t e through the secession scar or produce branches which soon secede as blastoconidia. Swelling and w a l l thickening occur i f f r e s h medium i s not supplied. 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APPENDIX I — MEDIA AND SOLUTIONS Defined Media (g/l) glucose + NO3 #1 (GN1) glucose 8.0 Ca(N03)2.4H20 0.5 MgS04.7H20 0.5 KH2P04 1.0 Conn's* 1.0 ml glucose + NO3 #6 (GN6) glucose 8.0 Ca(N03)2.4H20 4.0 MgS04.7H20 0.5 KH2P04 1.0 Conn's* 1.0 ml glucose + NO3 #5 (GN5) glucose 4.0 Ca(N03)2-4H20 2.0 MgS04.7H20 0.5 KH2P04 1.0 Conn's* 1.0 ml sucrose + ND3 sucrose 8.0 Ca(N03)2.4H20 4.0 MgS04.7H20 0.5 KH2P04 1.0 Conn's* 1.0 ml sorbose + NO3 sorbose 8.0 Ca(N03)2.4H20 4.0 MgS04.7H20 0.5 KH2PO4 1.0 Conn's* 1.0 ml citric acid + NO3** citric acid 9.3 Ca(N03)2.4H20 4.0 MgS04.7H20 0.5 KH2P04 1.0 Conn's* 1.0 ml glucose + glycine glucose 6.0 glycine 2.5 MgSO4.7H20 0.5 KH2P04 1.0 Conn's* 1.0 ml glucose + glutamic acid** glucose 2.9 glutamic acid 5.0 MgSO4.7H20 0.5 KH2P04 1.0 Conn's* 1.0 ml glucose + NH4 glucose + cystine glucose NH4C1 MgS04.7H20 KH2P04 Conn's* 8.0 1.8 0.5 1.0 1.0 ml glucose cystine MgS04.7H20 KH2P04 Conn's* 5.0 4.1 0.5 1.0 1.0 ml - 72 -glucose + asparagine glucose 4.0 asparagine 2.5 MgS04.7H20 0.5 KH 2P0 4 1.0 Conn's* 1.0 ml glucose + phenylalanine glucose 0.0 phenylalanine 5.6 MgSO4.7H20 0.5 KH 2P0 4 1.0 Conn's* 1.0 ml glucose + proline glucose 2.9 proline 3.9 MgS04.7H20 0.5 KH 2P0 4 1.0 Conn's* 1.0 ml * Conn's Trace Elements sterilized separately and added after autoclaving ** pH adjusted to 5.3 with NaOH Conn's Trace Elements d i s t i l l e d H 20 95.0 ml c i t r i c acid (monohydrate) 5.0 g ZnS04.7H20 5.0 g Fe(NIi4)2rS04)2.6H20 1.0 g Cu904 0.16 g MnS04.H20 0.05 g boric acid (anhydrous) 0.05 g Na2Mo04.2H20 0.05 g Malt-Yeast-Peptone (MYP) Phosphate Buffered Saline (pH 7.4) malt extract soytone yeast extract ICN agar d i s t i l l e d H 20 7.0 1.0 0.5 8.0 1.0 NaCl (1.0 M) KC1 (0.2 M) MgS04 (0.1 M) Na2HP04 (0.2 M) NaH2P04 (0.2 M) di s t i l l e d H20 12.0 ml 2.0 ml 1.0 ml 8.1 ml 1.9 ml 74.0 ml APPENDIX n — BATCH NUTRIENT STUDIES This appendix contains a series of graphs showing the extent of conidial branching over days 1 - 6 of batch growth on various media. Branch frequencies under the same conditions in GN6 medium are included with each histogram for easy comparison. Figure 1 — Growth on GNI medium. Figure 2 — Growth on sucrose + NO3 medium. Figure 3 — Growth on sorbose + NO3 medium. Figure 4 — Growth on c i t r i c acid + NO3 medium. Figure 5 — Growth on glucose + NH4 medium. Figure 6 — Growth on phenylalanine medium. Figure 7 — Growth on glucose + proline medium. Figure 8 — Growth on glucose + glycine medium. Figure 9 — Growth on glucose + cystine medium. Figure 10 — Growth on glucose + asparagine medium. Figure 11 — Growth on glucose + gluctamic acid medium. DAY 1 - 74 -BATCH GROWTH ON GNI TO-i DAY 2 l l l l BRANCHES/CONIDIUM DAY 3 Legend vza GNI mm GN6 l l l j l l l BRANCHES/CONIDIUM DAY 4 1. •V ^ S * S ^ * <k' BRANCHES / CONIDIUM o 2 2 t -S •.' «J *' -V «I <!>' ^>'^' BRANCHES/CONIDIUM DAY 5 DAY 6 1 I BRANCHES / CONIDIUM Figure 1 *>: v~»r «» *• -v * <ti •£ BRANCHES / CONIDIUM - 75 -BATCH GROWTH ON SUCROSE DAY 1 « S «> fc v • "V * » «p BRANCHCS/CONIOIUM DAY 2 VZZ S U C R O S E • • GN6 O N<v-b ». •> * -\ * "4 BRAMCHCS/CONIDIUU DAY 3 DAY 4 <S N T, *a *•*>*> "\tf «v 4> <J« BRANCHES/CONIDIUM BRANCHCS/CONIOIUM DAY 5 <a ^ <v fc •> * -\ t> «» ^ BRANCHCS/CONIOIUM Figure 2 o o M DAY 6 a ^ -v *>' fc * ti -\' t> ti «>' BRANCHCS/CONIOIUM - 76 -BATCH GROWTH ON SORBOSE DAY 1 O N -b fc •> *> 1 « « <p B R A N C H E S / C O N I D I U M DAY 2 Legend ^ SORBOSE Um GN6 ^—PL^ Lc^ a |L B R A N C H E S / C O N I D I U I DAY 3 DAY 4 B R A N C H E S / CONIDIUM O *> * A * H .0 ^ B R A N C H E S / C O N I D I U M DAY 5 O N «V k *> * •V « <»' <>' B R A N C H E S / CONIDIUM Figure 3 o o K DAY 6 B R A N C H E S / CONIDIUM - 77 -B A T C H G R O W T H O N C I T R I C A C I D DAY 1 DAY 2 BRANCHES / CONIDIUM Legend 2tt CITRIC ACID • • GN6 BRANCHCS/CONIOIUM DAY 3 DAY 4 ° N " V C *3 3 - V « 4 ,Qj< BRANCHCS/CONIOIUM .1 J j L o N T- -bS 3 - A 1 « « 4>' BRANCHCS/CONIDIUM DAY 5 O 40H o N -v -b w ^ *; -v «»• 4>' ^ BRANCHCS/CONIOIUM Figure 4 o M DAY 6 I L N *v -b * •> *; -v « <»• ^ BRANCHCS/CONIOIUM - 78 -BATCH GROWTH ON AMMONIUM DAY 1 B R A N C H C S / C O N I O I U M DAY 2 Legend ^ AMMONRJM mm GN6 O T. 1» V *> <> -\ «."»•*> B R A N C H C S / C O N I D I U M DAY 3 DAY 4 B R A N C H C S / C O N I D I U M B R A N C H C S / C O N I D I U M DAY 5 © N <v if W •}; *> A » * 4p> ^  B R A N C H C S / C O N I D I U M DAY 6 O ^ <V "V V ^ « -V « 4 j^t' B R A N C H C S / C O N I O I U M Figure 5 - 79 -B A T C H G R O W T H O N P H E N Y L A L A N I N E DAY 1 DAY 2 Legend PHENYLALANINE • • GN6 l l l I l 1,1 • • | BRANCHES/CONIDIUM "V "b ».•> «> A «<»4>^< BRANCHES/CONIDIUM DAY 3 DAY 4 BRANCHES/CONIDIUM BRANCHES / CONIDIUM DAY 5 DAY 6 ! 1 1 • I J J U < K «» w *>' B A" 5 <»' -p' BRANCHES / CONIDIUM Figure 6 ^ t> w v *> \ *> *' •«>; BRANCHES / CONIDIUM DAY 1 C> N ^ C *0 ft-V * * -P B R A N C H C S / C O N I D I U M - 80 -B A T C H G R O W T H O N P R O L I N E DAY 2 Legend VZ& PROLINE • • GN6 B R A N C H C S / C O N I D I U M DAY 3 DAY 4 S0-40-B R A N C H E S / C O N I D I U »o-40-* so-o o B R A N C H C S / C O N I O I U M DAY 5 I B R A N C H C S / C O N I D I U M Figure 7 DAY 6 •t *i > *£ *> -\' «i * *>' ^ B R A N C H C S / C O N I D I U M DAY 1 - 81 -B A T C H G R O W T H O N G L Y C I N E N S fc * > T «> "» •*>' BRANCHES/CONIDIUM DAY 2 Legend ^ GLYCINE •>• GN6 BRANCHES/CONIDIUM DAY 3 DAY 4 © \ A. A> fc *3 *> -V « <k «' BRANCHES / CONIDIUM 0 * > A . A > f c < 9 « > A » * .pi BRANCHES/CONIDIUM DAY 5 I F R—t , 1 , , , , R-© N "i " S S * > S A « * ^ BRANCHES / CONIDIUM o o M »o -DAY 6 -V *> W *> «> A » <» 4> BRANCHES/CONIDIUM Figure 8 - 82 -BATCH GROWTH ON CYSTINE DAY 1 -I <S N V i *> <b -\ 9> <» •£> BRANCHCS/CONIDIUM DAY 2 Legend EZ2 CYSTINE GN6 I 1 JJJJLi-l © 1. "b ». * » -\ t> "» *> ^  BRANCHCS/CONIDIUM DAY 3 2 \ W m. m - , O ^ "V "b b «j *> A t « ^  ^  BRANCHCS/CONIDIUM DAY 4 JUL O 1. "b * «» «> 'X ««4>>^< BRANCHCS/CONIDIUM DAY 5 KiLr^  ^-"f-"- , , , , S v *> -V * » <p BRANCHCS/CONIDIUM 4 - , DAY 6 Figure 9 ©"* *i -bfc * * -A « BRANCHCS/CONIDIUM - 83 -B A T C H G R O W T H O N A S P A R A G I N E DAY 1 DAY 2 H u l l - -^~ ir ~ ~ \ 4 * ^ B R A N C H E S / C O N I D I U M Legend ^ ASPARAGINE • • GN6 l i • • • • 1 • • - I B R A N C H E S / C O N I D I U M DAY 3 DAY 4 B R A N C H E S / C O N I D I U M O *>• A.A>• *3 » A * qi .0 ^p' B R A N C H E S / CONIDIUM DAY 5 DAY 6 I o I o O 40-1 *- "i "i v; •>: *. a * <» 45 «^ B R A N C H E S / C O N I D I U M o,_ " - i - "r[~ •b' "J" ^  «; -v « .0 < r' B R A N C H E S / CONIDIUM Figure 10 DAY 1 - 8 4 -BATCH GROWTH ON GLUTAMATE 9. 2 i l l i l l • P BRANCHCS/CONIDIUM DAY 2 GLUTAMATE H G N 6 BRANCHCS/CONIDIUM DAY 3 DAY 4 BRANCHES / CONIDIUM 2 I JJ I P ^ R p O N Tj ^  W <j to -V 5 « .f> j^f' BRANCHES / CONIDIUM DAY 5 <» "V "3 W *> *> A *> « 4p ^  BRANCHCS/CONIDIUM DAY 6 O M Figure 11 o •< X "SiW <»' *' -V «i « •*>' BRANCHES / CONIDIUM 

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