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Culture studies of an isolate of Saprolegnia Diclina Humphrey from Coho salmon, Onchorhynchus Kisutch… Chong, Shiow Ying 1973

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. CULTURE STUDIES OF AN ISOLATE OF SAPROLEGNIA DICLINA HUMPHREY FROM THE COHO SALMON, ONCORHYNCHUS KISUTCH (WALBAUM)  by SHIOW YING CHONG  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF BOTANY  We accept this thesis as conforming to the required standard  .THE UNIVERSITY OF BRITISH COLUMBIA 1973  In 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 of the r e q u i r e m e n t s f o r  an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the 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  I f u r t h e r agree 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 f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  study.  c o p y i n g of t h i s  be g r a n t e d by the Head of my  thesis  Department or  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 or p u b l i c a t i o n  of 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 not be a l l o w e d w i t h o u t written permission.  Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  my  ABSTRACT  Parasitic isolates of Saprolegnia diclina Humphrey from Coho salmon (Oncorhynchus kitsutch Walbaum) were grown under controlled laboratory conditions to test the effect of environmental parameters on growth. This study points out the significance of medium selection and methods of determining growth in introducing variability into the results of experimental studies of parasitic Saprolegniaceae. The results indicate that there is a significant effect of temperature and pH on growth and morphological development while the effects of phosphate concentration, aeration and nitrogen sources appear to be as significant but vary with medium used or method in determining growth.  ii  ACKNOWLEDGEMENT  My sincerest appreciation is extended to Dr. G.C. Hughes for his guidance and encouragement throughout this study and for his critical review on this manuscript. Thanks are also due to the following persons, either for their critical advice or for their generous assistance: Dr. R.J. Bandoni and Dr. J. Stein and Mr. G. Neish, Department of Botany, U.B. C. ; Mr. J. Baker, Robertson Creek Spawning Channel, Port Alberni, B.C.; Dr. T. Booth, Department of Botany, University of Manitoba; Mr. J.H. Mundie, Mr. J. McBride and Mr. A. L- Murray, Fisheries Research Board, Canada, in Vancouver; Mr. F. C. Boyd, Department of Fisheries and Forestry, Canada. I am also indebted to the Fisheries Research Board of Canada for support through a grant to Dr. Hughes and indebted to the University of British Columbia for a University Fellowship.  iii TABLE OF CONTENTS Page ABSTRACT  i  ACKNOWLEDGEMENT . .  ii  TABLE OF CONTENTS LIST OF TABLES, AND FIGURES  iii .  v  INTRODUCTION AND HISTORICAL RESUME  1  MATERIAL AND METHODS  7  I. Collection and Culture .. . . . Collection of fish mold Isolation, culture, decontamination technique and maintenance Examination of cultures Estimdionof growth Identification . .  7 7 . ••••  II. Experimental Work Culture studies Temperature Studies Growth effect of phosphate Growth effect of pH Growth effect of nitrogen sources Interpretation of data RESULTS  7 8 9 10 10 10 12 12 13 13 13  .  I. Collection and Culture Collection and identification On site examination of infected fish Identification of S. diclina II. Experimental Work Culture studies (1) Medium selection •••• (2) Effect of aeration The effect of temperature on growth and development of morphological structures The effect of KH^PO^- on growth and devel6pment of S. diclina •••• The effect of pH on the growth of S_. diclina .......  15 15 15 15 19 20 20 20 20 28 35 3.5  iv TABLE OF CONTENTS (Continued)  Page  The effect of nitrogen sources on the growth of Si. diclina  3  DISCUSSION SUMMARY  8  43 .  53  LITERATURE CITED  55  APPENDIX  ^3  V  LIST OF TABLES AND FIGURES TABLES  Page  I.  II.  III.  Fungi Collected from Infected Salmon, at Robertson Creek Spawning Channel, October, 1968 . .  17  Sequence and Timing of Development in Five Saprolegniaceous Fungi on Various Media at Approximately 25°C - r Summarized Results of Media Selection  21  Growth of S. diclina on Agar Blocks in Petri Dish Culture  22  Medium Selection of S. diclina on Various Media (Shaking in Psychotherm at 10°C)  23  Growth Study of S. diclima (dwt:mg) Under Different Aeration Regimes  24  Morphological Development of S. diclina at Different Temperatures on F13, MSM and BCHU  34  IV. V. VI. VII. VIII.  Effect of KtL^PO^ Concentration on Morphological Development of S. diclina (BCHU; 25°G; 2 weeks) ....  IX. X. XI.  XII.  18  The Result of pH Effect on Growth  37 38  Summarized Results in Availability of Nitrogen Sources for Growth of S. diclina Effect of Various Nitrogen Sources on Morphological Development of S. diclina on Modified Bhargava's Medium at 25°C (Standing)  40  Composition of Certain Natural Ingredients Used in the Media for Saprolegniaceous Fungi  46  Bubbling Apparatus  11  39  FIGURES 1  ... .  vi LIST OF TABLES AND FIGURES (Continued) FIGURES (Continued) .2 3 4  5 6 7 8 9 10 11 12 13  Page  Temperature of Robertson Creek (1967-1968)  16  Selection of Agar Block Inoculum for the Growth Study of S. diclina ..  25  Comparison of Different Aeration Regimes for Development of Structures Between F13 and BCHU Against Time  26  Results of Diameter Growth on PDA Under Different Temperature  29  Results of Diameter Growth on MSM Under Different Temperature  29  Results of Diameter Growth on BCHU Under Different Temperature .  30  Results of Diameter Growth on YpSs Under Different Temperature  30  Results of Diameter Growth on GYE Under Different Temperature  31  Results of Diameter Growth on F13 Under Different Temperature  31  Results of Temperature Studies ( dwt) on Three Different Media  32  Effect of KH_PO. on Growth 2 4 Effect of Nitrogen Sources on Growth  36 41  APPENDIX TABLES I. II.  MSM Medium (from McKay's Master Thesis, 1967) , Stock Solution for MSM Medium (from McKay's Master Thesis, 1967)  63 .  63  Vll  LIST OF TABLES AND FIGURES ( Continued) APPENDIX TABLES ( Continued) III. IV.  Ingredients of Media (BCHU, F13, GPA, GYE, Modified Bhargava's, PDA, YpSs) SPS Medium ( from Scott, Powell and Seymour, 1962)  Page  .  64 65  1 INTRODUCTION AND HISTORICAL RESUME Since the first paper mentioning fungal infection of fish (Aderon1748,in Ainsworth, 1958) and the earliest report of the Saproleg|  niaceae (Ledermiller, 1 760,in Honk, 1933) this group of fungi has been of great concern to fisheries biologists as a possible cause of fish mortality in North America (Rutter ,1904; Tiflhey ,1936, 1939; Rucker,1944; Hoffman, 1949 1963; Soott and O'Bier 1962), In England and Scotland (Brook 1879; Huxley1882 a, b), in the Netherlands and in a number of other countries, >  e.g. France, Germany and Russia. The f actors ..fOK fungal infections or* fish have been attributed to: (1) results of overcrowding, pollution and reduction of water supply (Brook 1879); (2) the results of temperature change (Cummin 1954, Scott and O'Bier 1962 and McKay, 1967); (3) nutritional deficiencies (Jewell^ Schneberger & Ross,1933); (4) mechanical injuries (e.g. 0 Doimell 194l; Hoffman^ 1949); ,  )  (5) the presence of a predisposing bacterial infection (Hardy, 1910:; Davis,, 1923; Rucker,, 1944; Anderson, 1972) or finally, (6) to the overall effect of combinations of the above factors. Rucker (1944) also showed that fish infections were related to the season. As well, Hughes (1962) reported that there was seasonal periodicity in occurrence and distribution of Saprolegniaceae. Seasonal change of the environment involves changes in water temperature, in light intensity, in oxygen, .in nutrient concentrations and in other environmental factors. The  2 environmental factors will simultaneously affect the physiological condition both of the fish and the fungi inhabiting the surrounding water. Since Klebs (1899) first stressed the importance of physiological studies, there have been few notable researches on the physiology or culture studies of Saprolegniaceous fungi, e. g. Kauffman (1908), Kanouse (1932), Volkonsky (1933), Wolf (1937) and Leonian and Lilly (1938). The studies of Saksena and Bhargava (1941) and Bhargava (1943,1945a,b,c) are more noteworthy than those of previous workers in that they conducted experiments under more carefully controlled conditions on chemically defined media. They also applied a quantitative approach to their results in the series of studies on the physiological aspects of Saprolegnia diclina Humphrey(sub• -S. delica Coker). However, there was no attempt to Standari'ze 1;nnoculum "quantitatively in their experiments. Other authors, e.g. Rucker (1944), Dayal (I960). Lee (1962), Willoughby (1962), Powell (1966), Gleason et al. (1970 a, b) and Powell et al. (1972) have completed similar studies in recent years. Reports on the physiological aspect of culture studies in Saprolegnia species seem to be numerous but complete and well organized studies on the subject are few.  The information we have  up to the present is so meager that Powell, Scott and Krieg (1972) have described S. parasitica as "virtually neglected" in this regard. Studies of growth, and the development of culturing techniques for saprolegniaceous fungi have been greatly influenced by several environmental parameters. Studies of temperature effects have resulted in dispute as to the actual role of temperature in growth of the fungi, in fish infection, and also as to the  3 optimum temperature for the structural development in different and in the same species. With the exception of Murray (1885) who stated that temperature was only of little importance except in extremes, most workers (Duff 1929,'. Saksena and Bhargava 1941; Rucker 1944; Lee, ,1962; Deverall , 1965 and Powell et al. ,1972) seem to favor the idea that temperature plays an important role in the growth and development of Saprolegniaceous fungi. Generally, growth rate is rapid at around 20°C to 28°C, decreases with a lowering of temperature and is inhibited with the increase of temperature above this range. However, in the natural environment, temperature is usually around 15°C and the original isolate used here was collected at 13°C in the field. The effects of pH on growth are rather complex, because an initial pH may affect early growth and subsequent growth will affect pH through the accumulation of metabolites in the environment. Studies on the effect of pH have also shown that the optimum range varies with species and also varies with medium and buffer used (Lee ,1962; Alexopoulos, 1966; Powell et al. 1972). Oxygen levels in experimental cultures have been found of interest in a number of studies in Saprolegniaceae. Duff (1929) concluded that the normal oxygen content of the water is sufficient for fungal growth in water culture. Saprolegniaceae are strict aerobes (Duff,1929; Rucker, 1944 and Alexopoulos 1966) and reproduction is inhibited in oxygen free water (Rucker 1944). However, saprolegniaceous zoospores are able to withstand a very low tension of oxygen for 72 hours while retaining their  4 power to germinate under more favorable conditions (Duff^l929). Powell (1966) confirmed Cochrane's (1958) view that shaking results in a significantly faster growth rate and found that shaking also supported highest dry weight yield in comparison to a stationary growth method. For sporangial formation and zoospores liberation, there is a very definite and consistent speeding up of the process when the material is well aerated (Cotner 1930). Gemmae, on the other hand, are usually produced in stationary cultures (Powell 1966). From most previous work, it would appear that increased oxygen usually enhances the growth, of Saprolegniaceae but that the formation of each morphological structure varies in its optimum oxygen requirement. Grove (1970) indicated that the major components of the fat free fraction of salmon are protein (40-60%), water, and ash. Saprolegniaceous fungi require a certain nutritionally available level of nitrogen, even though there are disagreements as to which nitrogen source is better utilized (Volkonsky, 1933; Leonian and Lilly, 1938; Bhargava, 1945; Whiffen,l945; Reischer,1951; Powell et al. 1972)_ A major problem confronting students of parasitic Saprolegniaceae has long been the scarcity of sexual reproductive structures in nature (Huxley 1882; Murray 1885) and the difficulty of inducing sexual reproduction in culture (Maurizio,1896; Kauffman ,1908; Scott and O'Bier 1962). Environmental factors which have been investigated in this regard include aeration ( Rucker, 1944), temperature (Duffjl929;* Lee1962; }  Szaniszlo,1965; Powell etal. 1972), pH (Duff,1929; Lee, 1962), light  5 (Duff,1929; Kanouse,1932; Lee,1962 and Szaniszlo1965) and general |  nutrition (Klebs,1899; Kanouse,1932; Wolf ,1937; Powell et al.,1972) . From most of'these studies, it ia seen that low temperature stimulates sexual reproduction and light affects oogonial formation to a certain degree. Among these factors, nutrition is the one which has been given a great deal of attention, following Kleb's (1899) early suggestion that reduction of food supply serves to induce sexual reproduction. The roles of phosphate, potassium, sodium and calcium levels on sexual formation and maturation in Saprolegniaceae (Kauffman^l908r Obel,1910; Pieters,l915; Kanouse^ 1932 and Wolf,1937) have all been investigated as the effects of levulose, maltose, glucose and peptone have (Pieters, 1915 and Kanouse, 1932). After Klebs'(1899) statement that the reduction of food supply induced sporulation, Pieters (1915) claimed that there was no constant relation between vegetative growth and sexual reproduction when the food offered exceeds the minimum concentration necessary for the given species in the Saprolegniaceae. However, Hawker (1966) gave the idea that these two phenomena have different nutritional requirements. The present study of parasitic isolates of Saprolegnia diclina from Coho salmon (Oncorhynchus kitsutch) was undertaken to provide data on cultivation under controlled laboratory conditions in an effort to broaden our knowledge of the effect of environmental factors on growth of this fungus and to assess the relationships of these results to the role of the test fungus as a fish parasite. It is hoped that this work will stimulate more interest in this type of research, demonstrate the desirability of careful  6 testing of the media used and elicit a better understanding of the variabilityintroduced in experimental studies of parasitic Saprolegniaceae through choice of culture media.  7 MATERIAL AND METHODS  I.  Collection and Culture Collection of fish molds Infected Coho and Sockeye (O. nerka Walbaum) were netted at  the water spill of the Robertson Creek Spawning Channel, Port Alberni, B.C. At the time offish collection, water temperature and pH were recorded on site. Small amounts of white fuzzy mycelium were picked off directly from the infected area of the fish with sterile tweezers and then transferred to PDA (Potato Dextrose Agar, Difco) plates or into a jar of sterile deionized water (100 ml) with hemp seed bait. The caps of the jars were loosened to allow sufficient aeration. General observations regarding the health of each fish, the fish size and weight; the extent, location and nature of the infection; and a brief description of each fungus examined in the field were recorded immediately after collection. Isolation, Culture, Decontamination Technique and Maintenance To get bacteria-free isolates, agar plugs with mycelial tips on two-day-old PDA cultures were cut with a #3 cork border and transferred to new PDA plates, with sterling silver rings inserted into the center (Raper, 1937; Powell & Tenny, 1964). Two days later, each of the agar blocks from this new PDA plate was transferred axenically to each sterile hempseed water culture and was grown at 20°C respectively. As soon as zoosporangia were mature they were cut off and each of them was placed in a sterile watch glass with sterile deionized water. Single spores were  8 picked up in .a sterile capillary tubeunderthe microscope. Each spore was inoculated in the center of a silver ring on a PDA plate to provide singlespore cultures for each isolate from where each stock culture was developed. Stock cultures were maintained on corn meal agar slants and oat meal agar . slants at 15°C and subcultured every six weeks. The inoculum blocks (6 mm diam. ) were always taken at 3 cm away from the center of culture plates for subculturing. Each kind of inoculum stock was developed on each medium selected for each experiment and transferred every seven days. Each culture was tested for presence of bacteria by subculturing in liquid maltose peptone and also by microscopic observation under phase contrast illumination before use. In order to secure more uniform results, all innoculations from inocular stocks to the experimental media and to the experimental water cultures were made from 72-hour subcultures at 20°C. Usually thirty inoculum blocks were obtained from the mycelial periphery 30 mm from the center of a 72-hour culture in a standard 100 mm petri dish. The height of agar blocks was controlled by pouring these plates with exactly 30 ml of agar medium each time. Examination of cultures Each liquid culture was transferred to a sterile glass petri dish (20 x 100mm) for examination of morphological structures. The observations were accomplished by inserting a plastic disc divided into five equal sectors in one of the eye pieces of a 15 x dissecting microscope. The relative occurrence frequency of each structure was recorded using  the symbols "+-" to 'M-+4-H-". This means of indicating frequency has little significance quantitatively but it does represent in a relative way, the morphological development of each colony. Any dubious frequencies were reconfirmed by staining or by examining the whole culture under a 100 x compound microscope. Estimation of growth Estimates of growth were conducted either by the measurement of colony diameter (five replicate  agar plates) or by determination of  the dry weight of the fungus growing on agar test blocks in water culture. The diameter growth was determined by modification of Testrake's method (1959), that growth was determined as the value of the mean of five separate means obtained from five random sets of two diameters, measured at right angles to one another, with the 6 mm  diameter of the original '  inoculum subtracted. Dry weight was determined at the end of each experimental incubation period. The value of the dry weight was calculated by subtracting the dry weight of ten control mycelial blocks from the gross dry weight of ten blocks from the experiment. Each of the ten control blocks was punched out between the ten experimental inoculum blocks. Each of the agar blocks, (control and experimental) was put on a pre-dried and pre-weighed 0.45 um Millipore filter disc in a pre-dried and preweighed foil cup and was in turn dried in a vacuum oven for 24 hours at 65°C with a pressure equivalent to 1381 mm mercury,.  The filter  discs were cooled in a vaseline sealed dessicator and weighed on a semianalytical Sartorious balance. The experimental blocks with the dispersed  10 spores, hyphal fragments and broken fungal pieces were collected on the Millipore filter disc in a Millipore filter funnel before they were put in the oven to dry.  Each culture flask was rinsed with sterile deionized water  into the filter funnel to assure that all fungal material had been recovered. Identification Identification of the fungi was based primarily on the characteristics of isolates growing on hempseed. Each culture was carefully checked with the description and figures in the major taxonomic works on the family: Humphrey (1893), Coker (1923), Fitzpatrick (1930), Coker & Mathews (1937), Johnson (1956), and Seymour (1970). Descriptions used here are mainly based upon the descriptive terminology of Johnson (1956). Fish species were identified from the keys and descriptions in Clemens and Willey (1961). II.  Experimental Work Culture Studies Experimental culture studies of S. diclina (isolate SYC #25) were  begun at room temperature (Table IV).  The presence of each structure on  each of a variety of experimental culture media was recorded every 24 hours, except for the first day when observations were made every 6 hours to see amy morphological structures that were produced earlier than 24 hours. In studies of medium selection for this isolate of S. diclina, the cultures were examined as indicated in Table II, IV and V. Medium selection in water cultures was determined by growing the fungi on blocks of the various  11 test media in 100 x 20 mm  glass petri-dishes containing 30 ml sterile  deionized water (pH 6.7). Each plate was examined every 12 hours from 12 hours up to 72 hours (Table IV). Diameter growth on agar plates, dry weight of the mycelium in water culture and the presence of any peculiar features were recorded at the end of the incubation period. For comparison of the growth under different conditions of aeration, the experiments designed to grow cultures under bubbling, shaking and stationary regimes using the range of experimental media for the inoculum blocks (Table VI, Fig. 3 and 4). The shaking speed of a New Brunwsick psychotherm incubator with reciprocating shaker was adjusted to 60 strokes/minute at 20°C. The bubbling apparatus was set up in a 20°C incubator and is as shown in Figure 1. Figure 1  Tygon Tube (120 cm; 5 mm  I. D. >—  Pasteur Dispo. Pipet Flask (125 ml) Sterile distilled H 0 (50 ml) 2  Tygon Tube (30 cm; 8 mm Plastic cap on the flask Cotton plug to filter out contaminants from air Ten 6 mm .diameter agar inocular  I. D. )  Six flasks were connected to the six outlets attached to a Marco aquarium air pump by tygon tubing and air was bubbled into the flasks through pasteur pipettes inserted in the flask cap to 6 mm below the medium. The regulatory valves were adjusted to provide approximately the same air flow into each flask. The top of each pipette had a small cotton plug inserted to act as a filter. Temperature Studies To remove the lag phase effect, each inoculum stock plate was kept for 2 hours before inoculation at the experimental temperature to be tested. Fraust 13 (low maltose and low peptone; Appendix Table VI), MSM (dextrose, vitamins, inorganic salts; Appendix Table V) and Booth and Chong's modified Harder, Uebelmesser and Fuller's medium (BCHU; high carbohydrate, high peptone and high yeast extract; Appendix Table VI) were chosen from the culture studies to grow the fungus at nine different temperatures (ranging from 0°C to 30°C) under a stationary aeration regime. Cultures were examined to determine presence and extent of development of reproductive structures. Dry weights were determined at the end of the incubation period of 2 weeks for each different medium used. The diameter growth on agar plates was recorded every 24 to 48 hours until maximum growth was attained or the experiment had run for its predetermined time. Growth Effects of Phosphate Five different molarities: from 0. 01 M to 0. 1M (Figure 12) of KH PO  were used as the buffering agent in  13 BCHU medium to test their effect on growth. pH was adjusted to 7 with 0. 2 N NaOH before autoclaving. Dry weights were determined at the end of two weeks incubation at 25°C, examination of morphological structures was also carried out at this time. Growth Effects of pH The effect of pH on growth was studied at six different pH's, ranging from 2. 5 to 9 (Table IX).  The medium (BCHU) was buffered with  0.05 M KH P0 and 0. 2 N NaOH and 0. 2 N HC*1 were used to adjust pH. 2  4  All the observations were made weekly and the dry weights were determined after two weeks under standing conditions at 25°C. Growth Effects of Nitrogen Sources Nine different nitrogen sources (Figure 13) were added respectively, in modified Bhargava s basal medium (Bhargava, 1.945b) to replace 1  his nitrogen source for these studies. The nitrogen sources were added at levels calculated to maintain a C:N ratio of 5:1. The extent that carbon was being supplied from the nitrogen sources was not determined. Each nitrogen source in the medium was increased by five times over the levels Bhargava (1945) used. Since the pH of the autoclaved deionized water prepared in our laboratory was 6. 7 to 6. 8, 0. 2 N NaOH was used to adjust the pH of each water culture at 7 before autoclaving. The experiments were carried out at 25°C. Dry weight and diameter growth were determined at the end of two weeks at which time morphological development was recorded. Interpretation of Data Statistical methods used in the interpretation of data are those  from Mitchell, Watson and Lipstran, (1963).  15 RESULTS I. . Collection and Culture Collection and identification The mean maximum temperature records for 1967 and 1968 the year of collection of fish and fungi - showed water temperatures between 12 °C and 14°C. The water temperature at the time of sampling at the dam and at the upstream station was 12°C. In 1967 and 1968, there was no dominant high temperature in the summer, the highest temperature being only 18°C to 21°C(Figure 2). pH of the water at the dam was 6.6 and at upstream, 5.8 at the time of collection of the infected salmon. The fungi found on the fish and isolated either from PDA plates or from hempseed baits in water culture are listed in Table I. Non-fruiting cultures of fungi were -keyed only.to genus as suggested by Scott (1964).  .  Se-  quence.and timing required for the development of various structures of different isolates on different kinds of media are shown in Table II. On Site Examination of Infected Fish The infected fish weighed from 1. 2kg to 2. 5kg.  The infection  sites on both Coho and Sockeye salmon were mainly on the dorsal surface from the head to the dorsal fin and on the caudal peduncle down to the caudal fin. This is more or less compatible with observations on sites of infection made by McKay (1967). Most of the infected fish found were dead, the live ones were sluggish and covered with big white fuzzy mycelial mats. Most of the infections involved only the epidermal layers but in a few instances, had developed into the body tissues and muscles  - A - mean lemp of 1967 - • - mean temp of 196'8  Figure 2. Temperature of Robertson Creek (1967-1968)  TABLE I Fungi Collected from Infected Salmon at Robertson Creek Spawning Channel October, 1968 Fungi  Achlya sp.  Host or Substrate sp. (Wt. of fish)  Infected Area of fish ,  Host Condition  O.nerka (1.68kg)  pectoral fin  dead  Associated with Gram negative bacteria and Isoachlya sp.  O.kisutchCl. 16kg)  dorsal part of head pectoral fin  dead dead  Associated with Gram negative bacteria and Protoachlya sp. Few associated with Achlya, Oogonia found once on 9 month old PDA culture  0.kisutch(2.45kg) Isoachlya sp.  0.nerka(l. 68kg)  Anal fin and Caudal fin  dead  Protoachlya parasitica Protoachlya sp. Pythium sp.  O.kisutchU. 16kg)  pelvic fin  dead  0.kisutch(l. 16kg)  pectoral fin  dead  salmon eggs, grass, sphagnum O.nerka (2.16kg) O.nerka (3.78kg)  head dorsal fin  dead dead  Saprolengia sp;  In the water sample of the surrounding area of dam Saprolegnia parasitica Saprolegnia diclina  Miscellaneous  O.kisutch (2. 07kg) O.kisutch (2. 21kg)  Gram negative bacteria along the hyphae Gram negative bacteria present Reproductive structure found on fish. Oogonium found in mycelium  dorsal fin. dorsal fin  living living  ted Gram negative bacteria associaGram negative bacteria associated  18  T A B L E II Sequence and T i m i n g of D e v e l o p m e n t i n S a p r o l e g n i a c e o u s o n V a r i o u s M e d i a at A p p r o x i m a t e l y 25°C  Medium  Days M y c e l i u m 1  Sporangium Oogonium  Antheridium  Fungi  Gemmae  S. d i c l i n a S. d i c l i n a  MSM  4 5  ti it  S. d i c l i n a (2)  6  ¥ 2 3 F13  4 5 6  A c h l y a sp # Achlya sp. S. d i c l i n a (1,2) S. p a r a s i t i c a S. d i c l i n a A c h l y a sp.  S. d i c l i n a (2)  S. d i c l i n a (1.2)  S. p a r a s i t i c a  S. d i c l i n a (1&2) Protoachlya  sp.  PDA  5 6  A c h l y a sp. A c h l y a s p . S. d i c l i n a (1) " Achlya sp. " S. d i c l i n a (2)  Achlya sp,  Protoachlya sp. 1 2 3 SPS  4 5 6 7  2 Hemp seed Water Culture  3  S. d i c l i n a (1.2)  S. p a r a s i t i c a P r o t o a c h l y a S. d i c l i n a Rrotoachlya S P(1&2) S. d i c l i n a S. d i c l i n a t>. d i c l i n a (1.2) at (1.2) 20 h r s ) S. d i c l i n a S . p a r a s i - S. p a r a s i tica tica Achlya s p > Protoachlya sp.  S.parasitica  4 5  7  A c h l y a sp_ S. d i c l i n a (2) P r o t o a c h l y a sp.  S. d i c l i n a S. p a r a s i t i c a  19 below the epidermis. Very few sexual reproductive structures of fungi were found on any of the infected fish and the infected salmon eggs in the surrounding water. A notable exception being the development of saprolegnian oogonia and antheridia on the head of a dead Sockeye (2. 16kg ) collected October, 1968. However, the fungi examined on the infected fish produced abundant zoosporangia. Gemmae were not observed at all. PDA proved to be the most satisfactory medium for initial isolations of these fish molds. The oligodynamic effect of the silver ring greatly facilitated the isolation of bacteria-free cultures and use of the rings was simple in practice. Single spore isolation was successful but proved to bea\slow and tedious process for preparing large numbers of cultures. Identification of S. diclina My #25 isolate was the principal experimental fungus of this study; it was collected in October, 1968 from the dorsal fin of a living Coho (2.21Kg) and identified as S. diclina.  The description of this species  follows: Saprolegnia diclina Humphrey, Trans. Amer. Phil. Soc. (N. S. ), 17: 109.pl. 17, Figs. 50-53. 1893. (See Seymour, 1970for synonymy.). ?  Vegetative hyphae: slender, diameter 12 pm - 30 pm, walls unconstricted; Gemmae: 22 pm - 55 pm, terminal, spherical to oval or intercalary and pyriform, occasionally irregular shape (30 x 150jam); Zoosporangia: abundant, 40 pm - 60 pm x 100jim.  -  400pm, clavate, straight with very obvious internal proliferation; Zoospore: 5. 5 pm x 10-12p.m, diplanetic, discharge saprolegnoid;  20 Oogonia: persistent, 50-70 um x 60 - 135^xm, sperical or pyriform, a few moniliform, most terminal or intercalary, a few in the emptied club-shaped zoo sporangium, pits 2.5^im to 5.5^am, present only where antheridia touch the oogonia; 5 14 oospheres (most 10 - 12); Antheridia: slender, diclinous, branching, not on every oogonium; Oospores: 8.5pxn  - 22^im,  spherical, not filling the oogonium, o'oplasm centric or subcentric. II. Experimental Work Culture Studies (1) Medium Selection The results of this study are presented in Tables II, IV, V and VI. Figure 3 and 4, involving growth and development on comparisons of a variety of media, are summarized in general terms in Table III. (2) Effect of Aeration In spite of culturing conditions, all morphological structures appeared in about two weeks on F13 medium (low carbohydrate and low nitrogen; Figure 4 and Table VI 3). Standing conditions appeared to be better for the development of antheridia and oogonia while under shaking conditions there was good growth of mycelium, but few sexual structures. The few oogonia seen were in chains with apically appressed antheridia as shown in Achlya sp. by Johnson (1956). On BCHU (Figure 4 and Table VI ;3 ) mycelium developed earlier than on F13 and antheridia were all distinctly dehiscent  21  TABLE III Summarized Results of Medium Selection  Medium Diam. growth  *BCHU  Hemp F13 GPA GYE Seed MSM PDA SPS YpSs 0  *** *** ***  Growth in dry weight Sporangia  0  ***  Sexual structure Gemmae Hyphal thickness  0  ****  ** * 5 ) e  **  ****:good; ***:moderate; **:fair; *:poor; 0:not tested *BCHU (high carbohydrate, hiqh peptone and high yeast extract) F13 (low maltose and peptone) GPA (glucose and peptone) GYE (glucose, yeast extract) HEMPSEED (protein, oil, resin narcotic) MSM (dextrose, vitamins, inorganic salts) PDA (potato, dextrose) SPS (glucose, organic nitrogen and inorganic salts) YpSs (yeast extract, phosphate, starch, sulfate) This result is concluded from all the data in this experiment, which are available for information of medium selection and most of the comparisons are done by student T-test.  TABLE IV Growth of S. diclina on Agar Blocks in Petri Pish Culture (Room Temperature 23°C - 28°C) (72 hrs; N = 5) Varieties of Agar Block Inoculum Examination Period 12 hrs. 24 hrs. 48 hrs. 72 hrs.  F13 *_  + +  Presence of wavy hyphae (mg)' dwt mean  PDA  -  -  -  Mean diameter Colony of mycelium (MM) 8(±0.89) Mean diameter of single hyphae  MSM  + +•  16(±1.09)  -  + +  GPA  + + + +  16  15  20  - •  -  -  -  Preferred medium based upon dwt, diameter 6 growth and condition of development  2  SPS  _  2.8(±0.06) 4  -  + +  26  3.00(to. 02) 3. 5  * + • presence; - absence ** N. A. not applicable  + + + 15.00(t0)  •-  3  Hemp Seed +•  + + +  13.20(^2.75) 16(t2. 75)  12  2. 00(t0) 3.2(to.4)  BCHU  1  18  43  -  -  2.2 5  5.7(*1.23) ** N. A. (control)  TABLE V (1) M e d i u m S e l e c t i o n o f S. d i c l i n a o n V a r i o u s M e d i a (Shaking i n P s y c h d h e r m at 1 0 ° C )  * D w t M e a n ( - 1 S. E.) m g  jN=5]___ Run No. ' l 8 t  1 week 2 weeks  \  5 days 1 week  2nd  ,  PDA 19.00( t 2.91) 19.40( 1=2.50)  GYE 3 4 . 20( X 4. 26) '. 3 8 . 0 0 ( ± 1.51)  YpSs 30. 60( t 4.83) 49.40(^5.12)  30.00( t 4.00) 3 0 . 4 0 ( t 7.22)  4 8 . 6 0 { ± 4.61) 64.00(t7.34)  66.20(t8.87) 65.60( t 5 t 55)  * Dwt : D r y Weight  (2) Morphological Development of S. diclina on Various media at 10 C, (Shaking) 1st Run 1 week 2 weeks  Medium  PDA  GYE  YpSs  Mycelium Sporangium Oogonium Anther idium Gemmae Oospore Mycelium Sporangium Oogonium Anther idium Gemmae Oospore Mycelium Sporangium Oogonium Anther idium Gemmae Oospore  + ++ +  + ++++  -  -  +  +  --  2nd Run 5 days  1 week  + ++ + +. +  + +++ ++ + +  -  -  +  + +  + mm  +  +  +  • +  -  -  -  -  + + .  -  -  —  ++++ —  ++ -  + + + • -  + -  -  -. •  -  -  TABLE VI  r  (1 jGrowth Study of S. diclina (*dwt:mg) Under Different Aeration Regimes (20°) 2 weeks (N = 3) Condition Standing Shaking Bubbling  4 weeks  6 weeks  (N = 6)  (N = 6) BCHU  F13  GYE  BCHU YpSs 34.33( to. 33) 14( to. 57) 10.66(±1.45) 33. 33(tl.76) 19.00(^2.88) 36 ( ±0.57)  21.66( ±2. 33) 15.66( ±0.88) 22.00( ±2.30)  39.16(±2.05) 47.83( *3.70) 61.83( t4.60)  25.16(±6.48) 46.50( ±3.92) 33.16( ±11.47)  * dwt : Dry weight (2) Morphological Development of S. diclina on Various MeuiS Under Different Conditions of Aeration (20°Cj 2 weeks) Condition  Stand• iig  YpSs 4 4+44  Mycelium Sporangium Oogonium Antheridium Gemmae Oospore .  Bubbling  GYE  4  .. 4 • ., 4+4+  ++ + :'•  +++ -  Mycelium Sporangium Shaking Oogonium Antheridium Gemmae Oospore  BCHU  -  4+  -.++  -  +  4  '+•' ++ •- •  4+4+ -• - •• +•  ^| | | |  •+  •' + •  Mycelium Sporangium Oogonium Antheridium Gemmae Oospore  ' *+" +++  4 1111  4+  4444+  «, .  4+  *• -  -  (3) Morphological Development of S. diclina on Var ious Media Under Different Conditions of Aeration (20°C) 3 4 weeks weeks F13 BCHU F13 F13 BCHU  1 week Condition •  Standing.  Mycelium Sporangium Oogonium Antheridium Gemmae Oospore  Mycelium Sporangium Shaking Oogonium Antheridium Gemmae Oospore _.  Bubblino  Mycelium Sporangium Oogonium Antheridium Gemmae Oospore  2 weeks  F13 +  .+  + +  +++ 4+++  +  ++++ +  +  4+ + ++  -  + + -  + + -  + -  *  + 4  + + +++ ' • .  + + + +  +  4 4+ +  + +++++  4+  •+ + ++ +. +++ ++: +++ + + + ' - ...+ + ++ + + •+ + +' + + + -  + +  4+ + +  4+ + + + + +  + . - = as in Table VII  •••  weeks BCHU  + '.'  ' + +  +  4  •+.  +  +  4  +  +  +  +  4 + +  '4 '. 4 4 4 4  4  4  4  +  +  •  +  -  •  '-  ' J  Temperature = . 23°-28" C N = 5 Incubation Period = 72 hrs v!\\'.i' dwt. mean mean diameter of single hyphae (10 readings each replicate) :  6 g  :  imean diameter growth of mycelium (4 readings each replicate)  £ ^ -  •4  Hempseed (Control)  BCHU  MSM  GPA  SPS  PDA  Figure 3. Selection of Agar Block ^Inoculum for the' Growth Study of S. diclina (from Table IV ) .  F13  N=6 Temperature=20°C -Frequency of sexual ^ structures Frequency of sporangia 100%  BCHU  F13  a) o £  0) s_  = 3 u o O  1  «*o  S> c QJ  cr <u s-  .i  »»  • *  iL  i  i i >  1  0%iWeeks 1234  234 1234  Shaking  234  Standing  Figure 4  iii 1234  II  234  Bubbling  J_L  246  •M i-LL  I! H  JJL  246 246 246 246 246  Shaking  Standing  Bubbling  Comparison of Different Aeration Regimes for Development of Structures between F 13 and BCHU against Time.(from Table VI,3) on  27  under all conditions of aeration. On BCHU, a few oogonia and antheridia appear in the fourth to sixth week with no exception of any culturing condition. However, sporangia appear earlier at the second week, especially under bubbling conditions. Two weeks appeared to be a sufficient time for the dry weight measurement of growth but not quite long enough for meaningful observations of sexual structures as compared with F13 for the same period. From Table VI (1), (2) and (3) and Fig. 4, it can also be seen that different media seem to have different optimal ranges of aeration for growth and for development of oogonia, antheridia, sporangia, gemmae, etc. At least in the first two weeks on YpSs, in the fourth week on F13 and in the sixth week on BCHU, aeration seems to affect the growth as determined by dry weight methods. Comparing ingredients (Appendix Table III), a medium (BCHU) with higher percentages of carbon and nitrogen seems to yield a higher dry weight than other media. The advantages of bubbling the agar blocks of BCHU did not show up until the sixth week. GYE agar blocks under standing and shaking produced numerous germinating zoospores in zoosporangia and the zoosporangia under shaking conditions were darker and smaller than those on BCHU or on YpSs. It is also notable that the sporangia on BCHU under shaking conditions had side-branching.  28 Oogonia and antheridia were scarcely found except a few on YpSs under bubbling and a few on BCHU under standing. Very few oospores were found in this experiment. Generally, standing supplies the best environment for formation of sexual structures while shaking and bubbling enhances the growth yield as measured by dry weight to a certain extent. Aeration, either by shaking or by bubbling, supports a little more sporangial production and much more hyphal growth over the longer period of incubation. The Effect of Temperature on Growth and Development of Morphological Structures From the diameter growth on the six different media, (Figures 5 to 10) it can be seen that there was no growth on the first day at 0°C regardless of which medium the fungus was on. The results can be divided approximately into four temperature ranges, (a) Between 20°C and 25°C growth was the best; (b) At 12-15°C, at 10°C and at 30°C the second-best; (c) At 3-4°C, 4-7°C and 7-8°C not too distinct and much below the linear growth line; (d) At 0°C, meager or none. With the exception of growth on F13 plates, there was generally good growth above 7-8°C and much poorer growth below this temperature. At 20°C and at 25°C growth on each medium plate reached the maximum diameter of 86 mm/plate by the third day. From the study of growth using dry weight measurement, (Figure 11) it is obvious that 0°C is the lower limit of growth when the fungus is on a medium with high (BCHU) or low (F13) percentage of carbon and nitrogen, but not necessarily the minimum limit for growth 6n a synthe-  A  0°C  e  3-4°C  •  4-7°C  A  7-8°C  •  IO°C  •  12-15°C  O  20°C  ©  25° C  +  30°C  Explanation of symbols used in Figs. 5-10.  1  2  3  4  5  Days Figure 5. Results of Diameter Growth on PDA Under Different Temperature * Symbols for each temperature - see p.  293'  * Symbols for each temperature - see p. 2^c* Figure 6. Results of Diameter Growth on MSM Under Different Temperature  6  30  Days * Symbols for each temperature - see p. 29a Figure 7. Results of Diameter Growth on BCHU Under Different Temperature  * Symbols for each temperature - sec p.  .29a  Figure 8. Results of Diameter Growth on YpSs Under Different Temperature  Days * Symbols for each temperature - see p.  '.  Figure 9. Results of Diameter Growth on GYE Under Different Temperature  * Symbols for each temperature - see p. 29a Figure 10. Results of Diameter Growth on F13 Under Different Temperature  32a  In this and all other figures, points represent: mean value with a vertical line showing a - 1 st. error.  Dwt. of Mycelium from 10 agar blocks (mg) :  O  tic medium like MSM. The optimum range of temperature for growth as measured by dry weight was between 22°C to 28°C, with the exception of the higher optimum at 30°C on BCHU. Examination of morphological features on all media (Table VII) showed that zoosporangial production reached its optimum from 7-8°C up to 30°C with the maximum number being produced in the second week. Zoospores were formed in the range from 10°C to 30°C but at 12-15°C, and at 20°C they were formed within 12 - 24 hours, as Duff (1929) had previously noted. There appeared to be no single optimum temperature for the formation of sexual structures on F13, BCHU, and MSM. However, on MSM, oogonia, antheridia and oospores were found at 25°C. My results indicate that it is almost impossible to demonstrate any direct relationship between temperature and formation of gemmae. In summary, MSM proved to be an adequate medium for studies of temperature effects on growth and on production and maturation of sexual organs. However, the differences in development of other morphological features at different temperatures was not very distinct, except for the formation of s zoospores. -I ^  .,  F13 proved to be poor for  this kind of temperature study, but the formation of sporangia and zoospore on this medium seems to be temperature-dependent. Growth of S. declina on BCHU yielded higher dry weights and larger numbers of gemmae and, as for MSM and F13, the development of sporangia and zoospores seemed temperature-dependent.  The symbol  no trace was found of the structure in question; "+": a distribution of one up to the area covering one fifth of the colony, with the exception for mycelium which "+ simply means "present"; M  "+++++" : indicates the presence of 200 to 250 or more oogonia, of 100 to 200 or more oospores, of 500 or more antheridia, of 500 or more sporangia, and of 250 or more gemmae, depending on which structure was being considered.  TABLE VII Morphological Development of S. diclina at Different Temperatures on F13. BCHU and MSM(2 weeks) Structure Mycelium  Sporangium  Oogonium  Anther idium  Oospore  Gemmae Zoospore Formation  Medium F13 BCHU MSM  o°c + +  4 -  -  F13 BCHU MSM  -  F13 BCHU MSM  -  F13 BCHU MSM  -  +  -  -  3-4°C 4 4 4 -  4-7°C 4 4 4 -  4 4-4-  4 4 -  +  -  +  +  +  -  -  +  4-4-4-  ++  4 -  4 -  +  -  -  4-4-  -  4-4-4-44-44 -  4-4-4-4-4-  4-4-4-  F13 BCHU MSM  4 4 4 -  4 4-4-4-44-4-  F13 BCHU MSM  -  -  -  4 -  4-4-  +  -  4 -  ++++ +++ ++ + ++ +  20°C 4 4 4 -  25°C 4 4 4 -  30°C 4 4 4 -  ++ +  4 4-4-44 -  4-44-44 -  4-44 4 -  +  4 -  4 4-4H4-4-  4-4-44 -  4 -  4 4 4-4-  4 > 4 -  4 -  4 -  + + +  ++  -  _  +++  4-4-4-  +  4 -  +4+  -  -  + + +  12-15°C  —  _  +  10°C  +  4-4+  +  -  +  ++ ++ +  F13 BCHU MSM  End of Hyphae F13 Swollen or BCHU Twisted MSM  7-8°C  +.  ++++  •H-+  +++  4-4-  4-4-4-4-  + +++ +  + +++ +  +  4 4-4-  4 4-4-  4 4 4 -  4 4 4 -  -  + + +  +44-+ 4 4 4 -  -  -  4 4 4-4-4-44-4-4+  4 4 -  35 The Effect of KP^PO^ on Growth and Development of S. diclina Phosphate levels (Figure 12) showed no effect on diameter growth despite the differences in molarities. However, at higher molarities, better growth was noted on BCHU in the dry weight determinations. Zoospores were released singly from the zoosporangia on media of 0.03 M KH^PO^. In cultures with 0. 07 M phosphate, the hyphae were highly branched. Development of reproductive structures (Table VIII) was inhibited at a concentration of 0.1 M even though this concentration yielded the highest dry weight and good diameter growth. Generally, concentration of the phosphates lower than 0. 05 M proved better for the development of sporangia and gemmae. The Effect of pH on the Growth of 5. diclina The results of pH studies on BCHU are given in Table IX. pH in the range of 5 to 8 supported fair growth and good development of sporangia. £>. diclina growing on BCHU buffered with 0.05 M phosphate seemed to have a wider optimum range and could tolerate the acidic pH's better than the basic ones. Oogonia and antheridia were not produced at the very acidic or very alkaline pH's, and formed only occasionally between pH 5 - 6.8.  The 0.05 M phosphate buffer seemed to inhibit oogonial  production (but not production of sporangia) even at the most favorable pH. Abnormal beaded hyphae, as reported by Lee (1962), were found at the extreme pH's, i.e., 2.5-3 and 9. Very few gemmae were found in this experiment except at pH 8 and pH 9.  .  "  36  4V  I I  i  20L35-.  I  I  15U  40 -  H  a  2C-  0 *-  0.01M  0.03M  0 20 40 Diam. Growth (mm)  0.05M 0.07M Concentration of KH,PO 2 4  0.1M  Figure 12. Effect of KH^PO^ ini [growth (Medium = BCHU ; 25°C; 2 weeks; N=9)  TABLE VIII .Effect of Phosphate Concentration on Morphological Development of S. diclina (BCHU; 25°C; 2 weeks) Structure Mycelium Sporangium  0.01 +  0.03 •  +  '  0.05  0.07  0.1  +  +•  +  ++  +•+•  +•  -  +++  +++  +++  +•  ++  ++  +++•  Oogonium Antheridium Gemmae Oospore Internal Proli feration of Zoosporium  * - +, +++  See Table VII  +  38 TABLE IX The Result of pH Effect on Growth at 25°C on BCHU Medium (2 weeks; buffered with 0. 05 KH P0 ) 2  pH  2.5  DWT (mg)  ' _3_  1 3.5 (±0.2) (-0.69)  _5_  6.8  8  25.13 (-2.22)  29.43 (±0.9-7)  17.76 (±1.04)  4  9 6.76 (±1.33)  Effect of Nitrogen Sources on the Growth of S. diclina The availability of nitrogen sotarces for growth of my isolate of S. diclina is as seen in Table X. Meanwhile, Figure 13 shows that culture growth on the same type of medium, using the same means of measuring growth, can give completely different results as a consequence of differences in culturing conditions. Statistical analyses of the results (Student's T-test) showed that in reaching the half-way-point to the maximum diameter growth  ... ammonium nitrate and tyrosine, as nitrogen  sources, supported faster growth rate in waiter culture; contrarily, Bactopeptone, histidine, L-asparagine, DL-asparatic acid, glutamic acid, control and valine supported faster growth rate on agar plates while dl-alanine yielded no difference of growth between growing on agar plate and in water culture. Unfortunately, one disadvantage of growing aquatic fungi on agar plates is that there is ;no chance to observe liberation of zoospores from the zoosporangia or the movement and activity of the primary and secondary zoospores. Hyphae growing on the agar blocks with ammonium nitrate in water culture were always thin, zigzag and wavy but abundant, with diameters of 4-6 to lOum, whereas with  TABLE X Summarized Results in Availability of Nitrogen Sources for Growth of S. diclina  DL A sparatic Acid  Ammonium Nitrate  L(+) glutamic L-aspaAcid ragine Control  Bactopeptone  LTyrosine  dlAlanine  Lvaline  Histidinei  Diameter growth on agar plate Diameter growth on agar block in water culture Dry weight yield on agar block in water culture  * "1" means the best source for growth, "2, 3 . . ...etc. " goes next in order. Grouping in these orders was done by pair-comparison through Student T Test .from data on fig.13.  TABLE XI Effect of Various Nitrogen Sources on Morphological Development of S. diclina On Modified Bhargava's Medium at 25°C, (standing) DL L(+) Aspa- Ammo- gluBacto- L- dlratic nium tamic L-aspapep- Tyro- AlaLAcid Nitrate Acid ragine Control tone sine nine valine 2 days old on agar plate (plate diam. 86 mm)  Mycelium Sporangium Oogonium Antheridium Gemmae  1 week old on agar block in water culture  Mycelium Sporangium Oogonium Antheridium Gemmae  + + ++++-  + + +  +  + • •H-+ ++•  + + +•+  +++  +•++  ++•+  Histidine  +•+  + + ++•  + + + +++++  , + - +++++ See Table VII  o  1  DL-Asparatic Acid  2  Ammonium Nitrate  3  L(+) Glutamic Acid  4  L-asparagine  5  Control  6  Bacto-peptone  7  L-Tyrosine  8  dl-Alanine  9  L-valine  10  Histidine  Explanation of numbers used in Fig. 13  50  5i-  BO*-  *  *  f  •  {  •  *  2!5r-  { • 10*30,-  10 _  10  oL I 0  I » Diam. 1 0 20  Growth (mm)  Figure 13.  Effect of Different Nitrogen Source on Growth  (Z5°CJ  on  MBM)  42 L-tyrosine in the medium hyphae were thick, 10-12 um.  dl-alanine and  DL-asparatic acid, were excellent nitrogen sources for the formation of gemmae while Bacto-peptone and L-asparagine only support fair numbers and one with L(+) glutamic acid, L-valine and histidine supported only few (Table XI). In water culture with L-asparagine mycelial growth was very poor, but irregularly-shaped gemmae were quite common. L(+) glutamic acid is a good nitrogen source supporting the development of sporangia while DL-asparatic acid, L-asparagine, Bacto-peptone, dl-alanine, support a few. Ammonium nitrate yielded good diameter growth in water culture and good dry weight growth while DL-arparatic acid also yielded good dry weight growth in water culture but good diameter growth on agar plate. \  Nevertheless, neither proved to be a good nitrogen source for supporting the development of reproductive structures. This probably means that good growth, in the usual sense of the term, does not always guarantee the best development of reproductive structures. It can also be seen (Figure 13) that the addition of the nitrogen sources to media with high carbon level (Modified Bhargava's) did not stimulate much better growth.  43  DISCUSSION The evaluation and comparison of the results of various studies of growth and development of the Saprolegniaceae have been complicated by several factors. They incltade different culturing methods, methods in estimation of growth, accuracy of identification and others. A few such factors which have been found interesting in my investigation are discussed here. Estimation of growth The dry weight method for the determination of growth and growth rate in Saprolegniaceae has been regarded as more accurate than other methods by most workers, (Bhargava,1945; Whiffen, 1945; Lilly and Barnett, 1951;  Reischer 195l; and Powell et al.,1972). However, re;  sults obtained by workers who have used this method are hard to compare since different ways of handling inoculum, use of different qualitative standards, and inadequate data on the different amounts of volatile substances present all affect the weighing results. My findings agree with those of Lilly arid Barnett (1951), and Mandel (1965) in that, in general, there is oftem little correlation between dry weight of fungi and diameter growth.-  -  ^-'..vU-  . This is primarily because  one is a measurement of growth In two dimensions, while the other is a three-dimensional measurement. It is also partially because dry weight data express in part, the accumulation of polysaccharides, lipids or wall material (Cochrane^ 1958) that may have little to do with the hyphal tip extension determinated by diameter growth measurements.  44 Throughout this study, cultures having greater diameter growth were found to have better morphological development. Whether the diameter growth and morphological development both reflect the vivacity of the protoplasmic function is unknown. Accordingly, if possible, both dry weight and diameter growth should be determined with the examination of morphological development throughout such studies. Media Contrary to most studies in which a single medium was used throughout a series of experiments (Bhargava,1945; Lee,1962; Powell et al.,1972). There was no single medium which proved best for all aspects investigated in this study. The choice of medium depends on the kind of infor/  mation sought and on a determination of the medium serving best for that purpose. Results indicate that development of reproductive structures and each physiological characteristic of S. diclina might have its own optimum physico-chemical conditions. Accordingly, comparisons of results between culture studies of the same fungus on different media have become more complicated than previous "single medium" studies indicate. The ingredients of media have also complicated the results since the same compound or same group of compounds, will react with other ingredients in the different media and have different effects on the same fungus. For instance, Teorell's buffer which was found generally satisfactory in the growth study of S. parasitica by Lee (1962) was reported by Powell et al. (1972) to be inhibitory to growth in the presence of the boric acid and sodium glutamate in their  45 standard medium. TRIS buffer was considered by Good (1966) as having poor buffering capacity below pH 7. 5, but was recommended by Powell et al. (1972) as superior in their medium. My finding that high levels of glucose and peptone in the medium (BCHU) yield better dry weight growth also demonstrates the importance of the concentration of the various ingredients in media used to study growth of aquatic Saprolegniaceae. Table XII summarizes information on the principal natural and semisynthetic components of culture media routinely used in such studies. It is pertinent to studies of the same fungus on different media and conversely suggests, to an extent, an explanation of the morphological differences noted in studies using the same medium. Culturing Condition From the results of my nitrogen studies, it appears that culturing conditions can alter the pattern of growth to a certain extent when other conditions are constant (Figure 13). However, no determinations were made of nitrogen levels in the agar used in the study. Some good growth under stationary conditions seems to support the finding of Duff (1929) that normal oxygen content of the water without additional bubbling is sufficient for fungal growth. Nevertheless, the results of shaking experiments in this investigation rejustify the point of view of Cochrane (1958) and Powell et al. (1972) and such agitation seems to enhance total dry weight yield more in the longer incubation period. Shaking does provide more homogenous environmental conditions and probably prolongs the phase of secondary growth which could be slowed after exhaustion of the original oxygen  46  T A B L E XII Composition of Certain Natural Ingredients Used in Media for Saprolegniaceous Fungi  Item (100 gr.)  Carbohydrate (sr.)  1  Corn  2  Hempseed  3  Lima Bean  68  Liver extract  +  4  20. 5  Oatmeal  68.2  7  Pea  17. 7  8  Peptone (Bacto)  9 Potato  Ca  6. 7  (trace of carbonate oxalate)  1.3  68  Mg  Na  P  381  120 and  7.5  (salts from barley)  7.4  54  5.2  365  0.4  22  1.9  122  16.16  0.058  0.27  0.00033  0. 22  0.056  1.08  0.22  1000 to 8000  2 to 8.2  37.4  JK  0. 5  20 •  14.2  Salmon extract (canned)  Fe  CI  8 (protein bodies)  19. 1  Yeast extract  Salts (Inorganic) (mg)  •1.2  20.7  50 to 70  6  11  3.7  Fat or Oil (gr.)  +  5 Malt extract  10  N(total) Protein (gr.) (gr.)  20.6  9.6  6.7  40  0.0016  106  0.29  6000 to 22.000 480  1.3  286  18.2  1893  Miscellaneous (mg) Organic and Vitamins (mg) 3 —  (I.NU) 390  680  0.15  0.14  0.6  0.24  0. 55  0. 14  0.36  0.18  B ^  B compi ex  Biotin  C  Enzymes  Folic Acid  12  Niacine  Choline  20  10  80  11  .Ether  Ash  0.37  3. 53  0. 14  0. 24  1.1 26  2. 1  0. 33 9  Resin Narcotic  17  480 6.5  9.69 to 43  0.04 or 5. 45  36. 2 to 213.6  1620  + = present but quantity unknown. Details - Bee Harrow and Mazur (1962), Malletle. Althouse and Clagett (1960), Claus and Tyler (1965), Merck Index (1908), Difco Manual (1969) and Trease and Evans (1972)  47  levels in the culture. '  However, on the matter for formation of  all reproductive structures, the nature of the medium seems to play a more important role than culturing conditions. Temperature Temperature was found to have significant effects on growth, the optimum range and cardinal points for each of the various media used are more or less compatible with ranges reported by workers who considered growth to be temperature-associated (Lee,1962; Powell et al. 1972). The Effect of Phosphate My findings are in agreement with Whiffen (1945) and Powell et al. (1972) as regards the effects of phosphate on development of reproductive structures, but not in regards to growth. The difference was probably due to the different media used and due to the higher buffering capacity of my phosphate concentrations. Saksena and Bhargava (1941) reported that S. diclina (sub. S, delica) failed to grow on their synthetic standard medium without phosphate (KH^PO^) and my isolate appears to grow better on media with higher molarity of phosphate. This supports the assumption that at lower levels, phosphate deficiency in the media causes lowered rates of glucose utilization  and other metabolic disturbances (Cochrane,1958).  The optimum pH range for growth of my isolate of £>. diclina, pH 5 to pH 8, is close to that reported by Duff (1929), Reischer (1951) and Powell_et al. (1972). It lies between Volkonsky's (1933) high and low optimum range but covers a wider range than Lee's (1962) pH 6.3 to pH 7.4, in spite of the different buffer used. Alexopoulus (1966) and Whiffen (1945) are the only workers to report an acidic pH optimum, from pH 4. 0 to pH 6. 0 for Saprolegnia spp. pH does not give a unitary effect because it affects enzymatic system, metal solubility, surface metabolic reaction, entry of essential vitamins, entry into the cell or organic acids or the uptake of minerals (Cochrane1958). The net 7  Change in pH is the result of interaction of changes in several processes including utilization of cations, utilization of amines, formation of acids from neutral metabolites (especially carbohydrates) and formation of bases (especially ammonia) from amino acids and proteins. The shift of pH occurs even in the presence of chelating agents to stabilize minerals because organic acids, e.g. pyruvic acid or succinic acid were always produced through hydrolysis of carbohydrates by fungi. Accumulation of these acids accounts for early depression of pH but later pH rises, following  the metabolic utilization of these acids  and nitrogen sources in the media. The amount of oxygen available to submerged mycelium is greater at acidic pH's (Lilly and Barnett,1951) and accordingly, respiratory rate at lower pH's should be greater.  *9 The above facts and assumptions probably explain, in part, why S. diclina has an optimum pH near neutral but is tolerant of pH's on the acidic side. Nitrogen Sources In my investigation of the effect of nitrogen sources on growth of S. diclina, the utilization of amino acids , proved similar to that reported by Powell et al7 (1972), but differ in regard to the utilization of histidine. Also, in my tests monoaminodicarboxylic acids and their amines appear to be  fairly good organic sources, as Volkonsky (1933) and  Bhargava (1945) noted. Utilization of amino acids depends on which amino acid is involved, i. e., primary or secondary, and also depends on which pathway being taken place by each fungal isolates. There are (a) deamination by hydrolysis, (b) deamination by hydrolysis, followed by decarboxylation, (c) oxidative deamination, (d) decarboxylation yielding amines, and (e) transamination. The results may reflect enzymatic permeabilities and capacities (Cochrane 1958) or merely be a secondary phenomenon resulting from pH changes during assimilation or it may be a metabolic consequence of the chelation of inorganic ions by amino acids. Additionally, amino acids and amides from natural sources may be contaminated with vitamins or they may also serve as a carbon source (Saksena and Bhargava^.941; Gleason1970a,b and Faro >  ;  1972.). Therefore, the attempt to elucidate the differences in assimilation of different amino acids by my isolate of S. diclina and the variation of results from other workers is not possible. Ammonium nitrate  50 acts as.a fairly good inorganic source in this study. It indicates, more likely, that ammonia is an effective nitrogen source for S. diclina as it is for S_. parasitica (Powell et al. 1972),  since most  workers (Hawker1950; Alexopoulos ,1966) report that nitrate is not ;  assimilated. Peptone (0.1%, Bacto) at the levels used in this study, did not prove to be a good nitrogen source with other nitrogen sources used. For certain unknown reasons, peptone in the modified Bhargava's medium was not assimilated and its presence in the medium neither promoted or inhibited the assimilation of the fungus on other ingredients. Development of morphological structures The effects of various environmental parameters on development of reproductive structures suggest that for morphological and reproductive development. Si. diclina probably needs some organic nitrogen sources (see under "ammonium nitrate" and "control". Table XL) Formation of sporangia and zoospore activity in these studies seemed to be affected by temperature (optimum 10°C - 25°C), aeration and pH (pH 5 - pH 8) beside food supply. In concurrence with Tiffney (1936)and Salvin (1942), I found that under low temperatures, differentiation of sporangial protoplasm failed to take place. The scarcity of discharge of zoospores in my culture may be partially attributed to the maintenance of culture at constant temperatures. Swollen and twisted hyphal tips found at temperatures between 3°C to 8°C and abnormal beaded hyphae at extreme pH's may indicate a response to unfavorable en-  51  vironmental conditions and under better conditions may become functional sporangia. For the production of gemmae, temperature and aeration do not seem to have any apparent enhancing effect. This is probably because there is no sudden reduction of food supply as suggested by Klebs (1899) and Hawker (1957), or because the nitrogen levels used were high enough to suppress their production. More likely, gemmae in my cultures function as resting structures produced in response to unfavorable environmental conditions .•.or. as a result of over-supply of nutrients (Kanouse 1932). Generally speaking, the formation of sexual structures on the test media occurred only in a very narrow temperature range, with the exception of MSM.  My find-  ings agree with those of most workers in that low temperatures favor sexual reproduction. However, production of sexual structures did not cease above 21°C in my cultures as Szaniszlo (1965) reported for £>. diclina. Induction of sexual reproduction may rely more on nutrient supply than on temperature. Other parameters reported to affect sexual reproduction are phosphate (DeBary, 1881; Klebs, 1899); nitrogen, potassium, sodium and calcium (Kauffman,1908); light and temperature (Szaniszlo, 1965) and probably some trace elements. The requirement for steroids and the effect of C:N ratio in the medium has been found to stimulate sexual reproduction for  JRyrhiviml anrl P T i y r n p K u  tho.ra and might also affect the formation and maturation of sexual structures in the Saprolegniaceae. A high C:N ratio was usually suggested as favoring oospore formation since low C:N ratios were  associated with the inhibitory pH rise resulting from accumulation of NH^-N derived from asparagine and other nitrogen sources. The C:N ratio in my nitrogen studies (C 5:N 1) proved to be too low to stimulate sexual reproduction because oogonia and oospores were not found frequently during this study. In my opinion, the lack of sexual reproduction on this S. diclina isolate in the laboratory and in the field is mainly a result of its narrow requirement of physico-chemical conditions. The differences noted in the laboratory in requirements of nitrogen for vegetative growth and for reproductive development, the narrow range of physical and chemical conditions promoting sexual reproduction as compared to vegetative growth, and the high occurrence of oogonia, oospores on MSM, more gemmae on MSM and abundance of gemmae on BCHU support this point of view. The evidence presented in this study emphasizes the importance that choice of culture media and methods of determining growth have on the outcome of laboratory studies of members of the Saprolegniaceae. These factors are of great significance if such laboratory studies are to provide meaningful data on nutrition and physiology of these fungi as well as elucidate their relationships to fish populations under natural conditions.  53 SUMMARY 1. Saprolegnia diclina Humphrey was isolated from infected Coho salmon (Oncorhynchus kitsutch) from Robertson Creek, B.C. 2.  Hempseed, BCHU, (Modified Booth and Chong's, Harder and Ubelmesser Media, high carbohydrate peptone and yeast extract) and MSM appear to be good media for experimental work while Potato Dextrose Agar is suitable for original isolation of fish molds.  3. Utilizing agar plugs of the various media as substrate total growth (as measured by dry weight) is low as compared to the other methods applied in other studies. 4. The lag phase of growth seems to be shorter on agar plates than in water culture. 5. Aeration by bubbling generally enhances the growth as measured by dry weight. 6. Temperatures between 22°C and 28°C, and pH between 5 and 8 are optimal for growth of this isolate of S. diclina. 7. Growth determined by dry weight and observed morphological development appear to be affected by the molarity of KH PC*while diameter growth is unaffected. 8. DL-asparatic acid, ammonium nitrate and L-glutamic acid are good sources of nitrogen for yield of dry weight in liquid culture while peptone, histidine and L-asparagine support good diameter growth on agar plates. Ammonium nitrate is indicated as the only good nitrogen source for diameter growth determinations in liquid culture.  54  Data indicate that there is big variation in the environmental and nutritional requirements for the development of vegetative structures and reproductive structures, especially sexual reproductive structures. The results also indicate the importance that the choice of culture media and the method of determining growth can assume in affecting the results of experimental studies of parasitic Saprolegniaceae.  §5 LITERATURE CITED Ainsworth, G. C. 1958. Fungous diseases of man and animals. New Biol. 27:51-69. Alexopoulos, C.J. 1966. Introductory mycology. 2nd ed. John Wiley and Sons, Inc., New York and London. 613 pp. Anderson, D.F. 1972. Virulence and persistence of rough and smooth form of Aeromonas salmonicida inoculated into Coho salmon (O. kitsutch). J. Fish. Res. Board Can. 29:204-206. Bary, A. de 1881.  Untersuchungentiberdie Peronosporeen und Saproleg-  nieen. Und die Etrundlagen eines nattfrlichen systems der Pilze. Beitr. Morph. Phys. der Pilze 4, 1-145. Bhargava, K. S. 1943. Physiological studies of some members of the family Saprolegniaceae. I. Enzyme action. J. Indian Bot. Soc. 12:85-99. .  1945a. Physiological studies on some members of the  family Saprolegniaceae. II. Sulphur and phosphorus requirements. Proc. Ind. Acad. Sci. 21:344-349. •.  . 1945b. Physiological studies on some members of the family Saprolegniaceae. III. Nitrogen requirements. J. Indian Bot. Soc. 24:67-72. . 1945c. Physiological studies on some members of the family Saprolegniaceae. IV. Carbohydrate requirements. Lloydia 8:60-68.  • .  *  56 Brook, G. 1879. Notes on the salmon disease in the Esk and Eden. Trans, and Proc. Bot. Soc. Edinburgh 13:389-394. Claus, E. and V. Tyler Jr. 1970. Pharmacognosy. 6th ed. Lea and FebigerCo., Baltimore. 518 pp. Clemens, W.A.  andG.V. Wiley. 1961. Fishes of the Pacific Coast of  Canada. 2nd ed. Fish. Res. Board of Can. Bull. Ottawa. Cochrane, V.W.  1958. Physiology of fungi. Wiley and Sons, Inc., New  York. 524 pp. Coker, W.C.  1923. The Saprolegniaceae. Univ. of N. Carolina Press,  Chapel Hill. 201 pp. and V. C. Mathews. 1937. Saprolegniales. North American Flora, 2, (l):15-67. \ •  \  Cotner, F.B. 1930. The development of the zoospores in the Oomycetes at optimum temperatures and the cytology of their active stages. Amer. J. Bot. 17:511-546. Cummin, R. 1954. Malachite-green oxalate used to control fungus on yellow pike perch eggs in jar hatchery operations. Prog. Fish Cult. 16:79-82. Davis, H.S. 1923. A new bacterial-disease of fresh water fishes. Bull. U.S. Bur. Fish 38:261-280. Dayal, R. I960. Utilization of a mixture of amino acids by some Saprolegniales. Proc. Nat. Acad. Sci., India, Sec. B. 29:49-52. Deverall, B.J. 1965. The physical environment for fungal growth I. Temperature, p. 543-550. In Ainsworth, G.C. and A.S. . Sussman (ed.). The Fungi, Vol. 1. Academic Press, New York.  57 Difco Manual of dehydrated culture media and reagents for microbiological and chemical laboratory procedures 9th ed., 1969. Difco Lab. In., Detroit, Mich. Duff, D. C.B.  1929. A physiological study of certain parasitic Saproleg-  niaceae. Contr. Can. Biol, and Fish., N.S., 5:195-202. Faro, S. 1972. Utilization of certain amino acid and carbohydrates as carbon sources by Achlya hetereosexualis. Mycologia 63:1234-1237. Fitzpatrick, H.M.  1930. Th e lower fungi:Phycomycetes.  McGraw-Hill  Book Co., New York. 331 pp. Gleason, F.H., C.R. Rudolph, and J.S. Price. 1972a. Growth of certain aquatic Oomycetes on amino acids. I. Saprolegnia, Achlya, Leptolegnia and Dictyuchus. Physiol. Plant. (Copenhagen) 23:513-516. , T.B. Stuart, J.S. Price, andT.T. Nelbach. 1970b. Growth of certain aquatic Oomycetes on amino acids.  II.  Apodachlya, Aphanomyces and Pythium Physiol. Plant. (Copenhagen) 23:769-774. Good. N. E., G.D. Winget, W  ;  Winter, T.N. Connolly, S. Izawa, and  R. Singh. 1966. Hydrogen ion buffers for biological research. Biochemistry:467-478. Groves, T.D.D. 1970. Body composition changes during growth in young sockeye (O. nerka) in fresh water. J. Fish. Res. Board Can. 27:929-942.  Harrow, B. and A. Mazur. 1962. Textbook of Biochemistry, 8th ed. Philadelphia,Saunders. 651 pp. Hardy, A. D.  1910. Association of alga and fungus in salmon disease .  Proc. Roy. Soc. Victoria 23(N.S. ):27-32. Hawker, L. E.  1950. Physiology of fungi. Oxford Univ. Press, London.  360 pp. .  1957. The physiology of reproduction in fungi. Cambridge  Univ. Press, London. 178 pp. .  1966. Environmental influences on reproduction, p. 435469. In Ainsworth, G . C. and A. S. Sussman (ed.) The Fungi, II. Academic Press, New York.  Hoffman, G . L i  1949. Isolation of Saprolegnia and Achlya with penicillin  and streptomycin and attempts to infect fish. Prog. Fish. Cult. 11:171-175. .  1963. Parasites of fresh water fish. I. Fungi of fish  and fish eggs. Fishery leaflet 564. U.S. Dept. of Int., Fish and Wildlife Service, Wash. D. C. 6 pp. Honk, W.  1933. Polyplanetism and zoospore germination in Saprolegnia and Pythium. Amer. J. Bot. 20:45-62.  Hughes, G . C.  1962. Seasonal periodicity of the Saprolegniaceae in the  southeastern United States. Trans. Brit. 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Saprolegnia mixta. Jahxb. f. wiss. Bot. 33:513-593. Lee, P.C., Jr. 1962. Some effect of pH, temperature and light on the production of zoosporangia in Saprolegnia parasitica Coker. M.A. Thesis, Univ. of Richmond, Virginia. Leonian, L. H. andV.G. Lilly. 1938. Studies on the nutrition of fungi. I. Thiamine, its constituents and the source of nitrogen. Phytopath. 28:531-548. Lilly, V. G. and Barnett, H. L.  1951. Physiology of the fungi. McGraw-  Hill Book Co., New York. 467 pp.  '60 Mallette, M.F., P.M. Althouse, and C. O. Clagett. I960. Biochemistry of plants and animals. John Wiley and Sons, Inc., New York. 552 pp. Mandel, G.R.  1965. Kinetics of fungal growth, p. 599-612. In Airisworth,  G. C. and A. S. Sussman, (ed.). The Fungi I, Academic Press, New York. . Maurizio, A. 1896. Studien Uber Saprolegnieen. Flora 82:14-31. McKay, D. 1967. Saprolegnia diclina Humphrey as a parasite of the salmonid Oncorhynchus kitsutch. M. Sc. Thesis. Univ. of British Columbia, Vancouver. Mitchell, C, Q.B. Watson, and O. Lipstran. 1963. Simplified Statistics. Powett Press Inc. Boulder, Colorado. 114 pp. Murray, G. 1885. Notes on the inoculation of fishes with Saprolegnia ferax. J. Bot. 23:302-308. Obel, P. 1910. Research on the conditions of the forming of oogonia in Achlya. Ann. Myc., 8:421-443. O'Donnell, D.J. 1941. A new method of combating fungus infection. Prog. Fish. Cult. 57:18-20. Pieters, A.J. 1915. The relation between vegetative vigor and reproduction in some Saprolegniaceae. Amer. J. Bot. 2:529-576. Powell, J.R. 1966. Some physiological aspects of growth and reproduction in Saprolegnia parasitica Coker. 128 p. Doctoral Dissertation .Virginia Polytechnic Inst., Blacksburg. and V. B. Tenny. 1964. Application of the oligodynamic effect to the separation of bacteria from Saprolegnia. Va. J. Sci. 15:298.  Powell, J.R-.., W.W.  Scott, and N.R. Krieg. 1972. Physiological  parameters of growth in Saprolegnia parasitica Coker. Mycopath. Mycol. Appl. 47:1-40. Raper, J.R. 1937. A method of freeing fungi from bacterial contamination. Science 85:342. Reischer, H.S. 1-951. Growth of Saprolegniaceae in synthetic medium. II. Nitrogen requirements and the role of Kleb's cycle acids. Mycologia 43:319-328. Rucker, R. R.  1944. A study of Saprolegnia infections among fish. 92 p.  Doctoral Dissertation, Univ. of Washington, Seattle. Rutter, C. 1904. Natural history of the quinnat salmon. A report of investigation in the Sacramento River, 1896-1901. Bull. U.S. Bur. Comm. Fish 22:65-142. Saksena, R. K. S. and K. S; Bhargava. 1941. A physiological study of Saprolegnia delica Coker. Proc. Nat. Acad. Sci., India, 11: 27-40. Salvin, S.B. 1941. Comparative studies on the primary and secondary zoospores of the Saprolegniaceae. I. Influence of temperature. Mycologia 33:592-600. Scott, W.W.  1964. Fungi associated with fish disease. Develop.  Ind.  Microbiol. 5:97-148. and A. H. O'Bier. 1962. Aquatic fungi associated with diseased fish and fish eggs. Prog. Fish. Cult. 24:3:15. J.R. Powell, and R.L. Seymour, 1963. Pure culture techniques applied to the growth of Saprolegnia spp. on a chemically defined medium. Va. J. Sci. 14:42-46.  62  Seymour, R. L. 1970. A revision of the Genus Saprolegnia. Nova Hedwigia 19:1-124. Stecher, P.G. and R.N.J. Merck. 1968. The Merck Index. 1641 pp. Szaniszlo, P.J. 1965. A study of the effect of light and temperature on the formation of oogonia and oospores in Saprolegnia diclina. J. Elisha Mitchell Sci. Soc. 81:10-15. TeStrake, D. 1959. Estuarine distribution and saline tolerance of Saprolegniaceae. Phyton, J. Exp. Bot. 12:147-152. Tiffney, W.W.  1936. A study of species of Saprolegnia attacking fish. Doc-  toral Dissertation, Harvard Univ. Cambridge, Mass. .  1939. The host range of Saprolegnia parasitica. Mycolo-  gia 31:310-321. Trease, G. E. and W. C. Evans. 1972. Pharmacognosy. 8th ed. Bailliere, Tindall, and Cassell, London. 795 pp. Volkonsky, M. 1933. Sur les conditions de sulture et le pouvoir de synthese de Saprolegnia sp. Ann. Inst. Pasteur., Paris, 50: 703-730. Whiffen, A. 1945. Nutritional studies of representatives of five genera in the Saprolegniaceae. J. Elisha Mitchell Sci. Soc. 61: 114-123. Willoughby, L. G. 1962. The occurrence and distribution of reproductive spores of Saprolegniales in fresh water. J. Ecol. 50:733-759. Wolf, F.T. 1937. A nutritional study of Achlya bisexualis and Saprolegnia } .  ferax. Amer. J. Bot. 24:119-123.  APPENDIX TABLE I . ( F r o m M c K a y 1967)  . Stock Solution9 for M S M  A P P E N D I X T A B L E \\ ( F r o m M c K a y 1967)  Medium MSM  Stock M i n e r a l Solution A: ' I,*.  CuSO 4 Fe(N0 3)3.9H20 . -. - M n S 0 4 . 4 H 2 0 ZnS04.7H20  .  0.4398 0.7235 0.203 .0.4398  g/1 » " "  2.  D i s s o l v e d ingredients f r o m step 1 i n 600 m l . d i s t i l l e d carbon* filtered water.  3.  Added enough concentrated H2SO4 to y i e l d a c l e a r solution.  4.  Made volume up to 1 l i t e r with w a t e r .  5.  Autoclaved at 15 lbs f o r 15 minutes; stored at 13°C.  6.  Used 2 m l . stock solution p e r l i t e r m e d i u m .  Stock M i n e r a l Solution B: 1.  •2.  CaCl2.2H20 CoCl2.6H20 H3B03 (NH4)5.Mo7024.7H20  0.05 0.02 0.02 0.02  g/1 " " "  Buffer: .  2. Inorganic nutrients:  Autoclaved at 15 lbs for 15 minutes; stored at 13°C.  4.  U s e d 1 m l . stock solution p e r l i t e r m e d i u m .  • Stock V i t a m i n Solution: D-.Biotin I-Inositol Nicotinuric acid P - A m i n o b e n z o i c acid Pantothenol Pyridoxine-HCl Riboflavin Thiamine-HCl  0.001 g/1 1.0 " 0. 2 " 0.02 " 0 . 1 " 0,02 " 0 . 1 " 0.02 "  2.  D i s s o l v e d ingredients f r o m step 1 in 400 m l . 2 0 % ethyl a l c o h o l .  3.  Stored at r o o m  4.  Used 2 m l 6tock solution p e r l i t e r m e d i u m .  temperature.  CaCl2.6H20  0.005  • n  CoCl2.6H20  0.002  11  CuSO. 4 Fe{N03)3.9H20  0.0002  11  0.0002  11  H3B03  0.002  11  MnS04.4H20  0.0001  11  NaCl  6.01  11  ZnS04.7H20  3.  Organic nutrients:  g/1  KH2P04  .(NH 4 ) 6 M o 7 0 2 4 . 7 H 2 0  D i s s o l v e d ingredients f r o m step 1 i n 100 m l . d i s t i l l e d carbonfiltered water.  3.  1.  1.  Medium  C a s e i n hydrolysate Dextrose Biotin Inositol Nicotinuric acid P-Aminobenzoic acid Pantothenol Pyridoxine-HCl ( . Riboflavin Thiamine  0.002 0.0002 1.5 2.0 0.005 5.0 1.0 0.1 0.5 0.1 0. 5 0.1 20.0  II II  mg/l  II 11  11 •  g/1 1  4.  A g a r (for s o l i d m e d i a only)  5.  D i s t i l l e d c a r b o n - f i l t e r e d water  6.  D i s s o l v e d K H 2 P 0 4 . c a s e i n hydrolsate. N a C l , and agar i n 900 m l water i n 1 1. f l a s k with detachable 25 m l d i s p e n s e r . Using s t e r i l e pipettes, added 2 m l stock m i n e r a l solution and 1 m l stock m i n e r a l solution B .  7.  D i s s o l v e d dextrose i n 100 m l . water i n separate E r h l e n m e y e r f l a s k .  8.  Autoclaved flasks f r o m steps 6 and 7 at 15 lbs for 15 m i n u t e s .  9.  Added dextrose solution to hot m e d i u m .  10.  1.0  Using s t e r i l e pipette, added 2 m l stock v i t a m i n solution to s l i g h t l y cooled m e d i u m .  cr>  64  APPENDIX TABLE III Ingredients of Media  Ingredients gr /I 000 ml  *BCHU  F13  GPA  Agar (Bacto)  20  20  15  Dist.HO(ml) Dextrpse(Glucose)  1000 ml  Media GYE 20  1.2  PDA  20  15  00 ml  1000 ml 1000 ml  10  MBM  10  10  20  1000 ml 1000 ml 20  0. 5  KH FO 2 4 Liver extract  2.5  Malt extract  15  1  1.5  Maltose  0.5  MgS0  4  MgCl .6H 0  0. 5  Na S 2 Peptone (Bacto)  0. 17  2  YpSs  2  15  0.4  1.0  Potato Starch  200 5  15  Yeast extract  4  *BCHU (Booth and Chong's modification of Harder, Uebelmesser and. Fuller's medium. F13 = Fraust 13. GYE  = Glucose yeast extract agar.  GPA = Glucose peptone agar, see Kanouse 1932. PDA = Potato Dextrose Agar (Bacto).  YpSs = Emerson YpSs agar. MBM  = Modified Bhargava's medium for control in nitrogen studies. Nitrogen sources were added to supply C:N = 5:1.  65  APPENDIX TABLE IV SPS MEDIUM (From Scott, Powell and Seymour 1963)  1. Chelation agent: Ethylenediaminetetraacetic acid  0.5  2. Buffer for pH 7. 0: K HP0  0. 17  2  4  KH.PC> 2 4  grams/liter  0. 14  3. Inorganic nutrients: MgCl . 6H O  1.0  CaCl .6 H O  0.02  MnCl .4H 0  0.06  ZnCl .  0.04 0.0013  z  2  z  2  2  2  FeCl .6H 0 3  2  4. Organic Nutrients: DL Methionine Glucose Sodium glutamate (mono)  0.05 5. 0 2.0  5. Dissolve the ingredients of steps 1, 2, 3 and 4 in 971 ml. predistilled ion-exchanged water and adjust the pH of the solution to 7. 0 with KOH pellets. 6. Agar (Difco Bacto-agar) Add to the above solution.  20.0  7. Autoclave the medium at 1 5 lbs for 30 minutes.  11  

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