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A study of the physiology and strains of Ophiostoma fimbriatum (E&H) Nann Madhosing, Clarence 1957

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A STUDY OF THE PHYSIOLOGY AND STRAINS OF OPHIOSTOMA FIMBRIATUM (E & H) NAM. by CLARENCE MADHOSINGH B.S.A., University of B r i t i s h Columbia, 1954  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS •in the Department of Botany and Biology  We accept t h i s thesis as conforming t o the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1957  - i  -  ABSTRACT  The fungus Ophiostoma fimbriatum (E & H) Nann, though exceptiona l l y rare i n northern climates, i s f a i r l y widespread i n t r o p i c a l and sub-tropical areas of the world causing diseases on many species of p l a n t s . The disease producing c a p a b i l i t i e s of the fungus have become a major economic problem i n the growth and storage of sweet potatoes (Ipomoea batatas ( L . ) Lam) i n the southern parts of the United States* The organism i s i n t e r e s t i n g from the point of view that  it  produces, very r e a d i l y on sweet potate dextrose agar, two types of ; asexual or vegetative spores and the perfect stage with the p e r i t h e c i a containing ascospores.  Several strains of the fungi have been i s o l a t e d from natural  habitats. This work deals, i n general, with a study of the gross morphology of t h i s ascomycete and some observations on the nuclear apparatus of the resting and germinating c o n i d i a .  More s p e c i f i c a l l y , t h i s study t r e a t s  with c e r t a i n factors i n n u t r i t i o n which affect the physiology i n such a way that the growth and sporulation c h a r a c t e r i s t i c s of the organism are altered. Since several strains of 0 . fimbriatum have been i s o l a t e d n a t u r a l l y i t i s thought that these must have been derived from mutant changes occurring i n an o r i g i n a l "wild" form which was propagated to more susceptible v a r i e t a l hosts.  As a r e s u l t , studies are undertaken i n an  attempt to induce changes i n an o r i g i n a l culture by adopting a r t i f i c i a l mutagenic methods.  A pathogenicity experiment i s done on sweet potato  blocks i n the laboratory to a s c e r t a i n the r e l a t i v e degree of virulence between the new-formed s t r a i n s .  - ii » This work shows that the c u l t u r a l c h a r a c t e r i s t i c s and r e p r o ductive behaviour of t h i s fungus could be modified by s p e c i f i c i n the culture medium.  variations  It i s shown among other things, that copper, i n  the role of a micro-nutrient, plays a d e f i n i t e part i n the  manifestation  of sexuality and i n the development of pigmentation i n the organism. "Mutations" are produced by using X - i r r i d i a t i o n and u l t r a - v i o l e t rays as inductive agents.  Many of the new-formed "mutants" are unstable  and back mutation to the o r i g i n a l "wild" type i s common.  In presenting the  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  r e q u i r e m e n t s f o r an advanced degree at the  University  o f 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 agree t h a t p e r m i s s i o n for. e x t e n s i v e f o r s c h o l a r l y purposes may  study.  I further  copying of t h i s  be g r a n t e d by the Head o f  Department o r by h i s r e p r e s e n t a t i v e .  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 my w r i t t e n  Department o f  &  The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver S, Canada. Date  QdjLptt^&j^  l9  f  my  I t i s understood  that copying 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  ^tgy^Aj n^yvd fcjjL  thesis  financial  permission.  - iii TABLE OF CONTENTS Page Abstract  i  Table of Contents  iii  Acknowledgments  iv 1  Review of L i t e r a t u r e (i) Classification  1  ( i i ) Sexuality and Strains  2  A Morphological Study  8 13  A Cytological Study Physiological and Mutagenic Studies The effect of thiamine hydrochloride on growth and sporulation  *  17  The effect of colchicine on growth and sporulation  20  The effect of camphor on growth and sporulation  26  The effect of various sources of nitrogen on the growth sporulation  30  The effect of copper on growth  34  The induction of mutations by X-rays and u l t r a - v i o l e t rays  41  Further I r r a d i a t i o n Studies The effect of u l t r a - v i o l e t rays on spores  54 59  Pathogenicity Study  62  Appendix".'..^  6g  Bibliography  76  - iv -  ACKNOWLEDGMENTS In the compilation of t h i s work, I have received h e l p f u l advice and assistance from various persons to whom I wish to convey my gratitude.  F i r s t l y , my thanks go to my professor, Dr. Frank Dickson,  of the Department of Botany a t t h i s University f o r h i s help and patient guidance and also to the l a t e Dr. D . C . Buckland f o r the use of the equipment and materials i n his Forest Pathology Laboratory f o r the accomplishment of a part of t h i s work. Thanks to D r . J . S . L o t t , R a d i o l o g i s t , and Mr. Maurice B u l l e n , Research P h y s i c i s t , both of the Ontario Cancer C l i n i c (London Branch) for making available the f a c i l i t i e s  of the X-ray machine used for the  induction of mutations* I also extend my gratitude to Dr. W . J . Martin, Associate Plant Pathologist at the Louisiana A g r i c u l t u r a l Experimental Station at Baton Rouge for the i s o l a t e CeratostomeHa fimbriata B , which was used extens i v e l y i n t h i s study.  Thanks too, to Miss E l l e n Sprinnget for her  technical help and for the typing of t h i s t h e s i s .  REVIEW OF LITERATURE  (i)  Classification Ceratocystis fimbriatum E l l & Hals Sphaeronema fimbriatum  ( E l l & Hals) Sacc,  Ceratostomellafdmbriata ( E l l & Hals) E H . Ophiostoma fimbriatum  ( E l l & Hals) Nann.  The disease was f i r s t described i n 1890 by Halsted (1), who gave the fungus the name Ceratocystis fimbriatum.  Owing to the early-  d i s i n t e g r a t i o n of the ascus walls and the l i b e r a t i o n of the ascospores into the cavity of the mature p e r i t h e c i a , Saccajrdo (2) regarded the fkuo&ing body as a pycnidium and transferred the organism to the form genus Sphaeronema of the Fungi Imperfecti i n 1892. Stevens, i n 1913 (3),  described the fungus as having globose  pycnidia 100-200yu i n diameter surrounded by septate hyaline hyphae. pycnidia possessed a rostrum 20-30^ long and a p i c a l l y fimbriate. found that the conidia were g l o b o s e - e l l i p t i c  5-9^  long.  The He  The fungus grew  well on sweet potato producing dark, almost black, spots on the s k i n . In a r t i f i c i a l culture the mycelium was dark, abundantly septate and with numerous o i l globules.  Long multiseptate conidiophores with l i g h t  t i p s arose from the medium.  colored  From these, Stevens thought, hyaline conidia  were produced endogenously. Lehman (4) i n 1918 distinguished two types of conidia i n Sphaeronema fimbriatum. conidia".  These he termed "hyaline conidia" and "olive  The "olive" conidia were described as being produced exogenously  while the "hyaline" conidia were produced endogenously.  - 2 -  In 1923 Elliott (5) recognized the perfect stage and placed the fungus i n Ceratostomella fimbriata i n the Ceratostomatacae of the Sphaeriales. In 1932 Nannfeldt (6) removed the fungus from the Ceratostomatacae and placed i t i n the Ophiostomatacae.  These two families are very  similar except that Nannfeldt reserves the Ophiostomatacae for fungi whose asci are not accompanied by paraphyses and whose asci walls undergo autodigestion. Richard and Olga Falck (7) i n 1947 created another "class" of the "Ascomycetales" which they called "Class Haerangiomycetes".  In this  class they placed Ophiostoma and Melanospora i n which the asci do not possess a definite c e l l wall but merely a plasma membrane or where the ascus wall i s almost immediately dissolved after i t s formation.  In these  fungi, therefore, the ascus does not exercise i t s normal function of ascospore dispersion but these spores are carried out through the ostiole in a mass of "mucus" and rest i n a drop i n the funnel-like "haerangium" formed by filaments diverging from the edge of the ostiole.  This "class"  the authors regard as an evolutionary development from Sphaeriales i n which definite functional asci occur. ( i i ) Sexuality and Strains E l l i o t t (8) made a cytological study of the fungus and found that throughout the vegetative state the sweet potato black rot fungus was uninucleate as was seen i n the hyphal cells and.both'forms of the asexual spores.  He found also, that two hyphal branches formed a knot which  developed into a perithecium.  whether this i s due to different asexual  elements i s not certain but i n favour of this, E l l i o t t found that perithecia  - 3 were always formed i n groups, usually crowded together i n narrow zones and without relation to the abundance of mycelium or of the asexual fruiting stages.  The hyphal branch which was to become the ascogonium  thickened at the base while the antheridial branch twisted around i t . The ascogonium differentiated into a basal c e l l , an ascogonium, and a trichogyne.  The trichogyne was continuous with the ascogonium; there was  no dividing c e l l wall and this structure had no nucleus.  The ascogonial  nucleus, at this time, was very prominent. The trichogyne and fused at whatever point they came i n contact with one another.  antheridium The  uninucleate upper c e l l made an opening and liberated the male nucleus into the trichogyne.  The nucleus passed downward enlarging as i t went  and proceeded to the body of the oogone. As a result of the numerous nuclear divisions occurring conjugately many pairs of nuclei arose which passed out into non-septate ascogenous hyphae. In the meantime, the cells under the ascogonium had developed to form a closed perithecium with a dark-colored outer wall and thin-walled hyaline inner cells to which the ascogenous hyphae became attached and developed i n the perithecial cavity.  Eight ascospores arose i n each ascus, and ascus walls, ascogenous  hyphae and the remainder of the 'nurse c e l l s ' underwent autodigestion. Gertrud Mittman i n 1932 to find any antherid.  (9) contrary to E l l i o t t ' s report, failed  According to her, the ascogonium began as a single,  uninucleate, somewhat curved terminal c e l l of a short lateral branch. This c e l l elongated, coiled and divided into three to five uninucleate ascogonial cells which enlarged and differentiated from the enveloping perithecial wall cells, and developed into mature asci.  Since Miss Mittmann  did not report any fusion of sexual nuclei this may indicate that somatogamy had taken place between hyphal cells previously.  - 4 Andrus and Harter (10), i n contradiction of E l l i o t t , that i t was  thought  strange that the oogonium should display such intimate contact  with the antheridium f o r apparently a f u s i o n of the two structures r a r e l y i f ever occurred.  In two instances they observed a binucleate terminal  c e l l which suggested to them that the terminal c e l l acted as s trichogyne. They have never, however, noticed any conjugation.  These workers proposed  the idea that the oogonium was probably f e r t i l i z e d by a nucleus from the terminal c e l l i n a manner similar to that described by F a u l l f o r Laboulbenia chaetophora Thax. (11). Andrus and Harter further stated that the oogonium by i t s d i v i s i o n and by further d i v i s i o n of i t s progeny, formed a multitude of c e l l s a l l destined to become a s c i .  They observed the complete absence  of any hyphal connection between the body of the perithecium and the ascogenous c e l l s which occupy i t s i n t e r i o r .  They thought t h i s s i t u a t i o n  occurred because these c e l l s were not derived from ascogenous hyphae but from a progressive c e l l f i s s i o n that began with the o r i g i n a l protoplast i n the sub-terminal c e l l of the ascogone.  These c e l l s were imbedded i n a  nutrient medium which obviated the need f o r connecting hyphae. Andrus and Harter (10) confirmed Mittman as to the absence of croziers and as to the unwalled condition of the a s c i i n C. fimbriata. They further reported that the f i r s t nuclear d i v i s i o n i s characterised by the development of a d i s t i n c t ascus inside which, they suggested, seemed to be the membrane of the f u s i o n nucleus.  They considered that a l l three  nuclear d i v i s i o n s occurred w i t h i n t h i s v e s i c l e .  Andrus and Harter further  maintained that t h i s v e s i c l e expanded during nuclear d i v i s i o n and, by the time the spores were formed, became the wall of the ascus.  These are  the f i r s t workers to report the membrane of a fusion nucleus becoming the  - 5 wall of an ascus.  They suggested that "the procedure i s  doubtless  peculiar to those species of Pyrenomycetes whose a s c i i n t h e i r younger stages are without a d e f i n i t e w a l l " . Later work by Andrus and Harter (12) i n 1937, confirmed t h e i r e a r l i e r views and they further concluded that i n the i n i t i a t i o n of the a s c i , not only d i r e c t and i n d i r e c t types of cleavage occurred, but also that the t y p i c a l c r o z i e r type of cleavage might have taken place.  They  found that the fusion nucleus consisted of a chromatin network with a nucleolus.  In the f i r s t d i v i s i o n two comma-shaped and two bilobed bodies  were found.  These authors reported no reduction d i v i s i o n i n the ascus.  Gwynne-Vaughan and Broadhead (13) observed a t y p i c a l ascomycetous development of the f r u i t i n g body, contrary to the findings of Andrus and Harter (10).  They f a i l e d to confirm the absence of ascogenous  hyphae and ascus w a l l s , as reported by Andrus and Harter and could f i n d no evidence of the ascus vesicle figured by Andrus and Harter and Mittmann i n the ascus.  They found that the a s c i developed by t y p i c a l c r o z i e r  formation. Mittmann, i n 1932, demonstrated the homothallic nature of Geratostomella fimbriata.  Olson, however, i n 1950 (14) succeeded i n  i s o l a t i n g h e t e r o t h a l l i c strains of t h i s organism. Gaumann and Dodge (15) stated that there seemed to be a s l i g h t suggestion of heterothallism since antheridia and oogonia arose from separate hyphal strands.  The antheridium c o i l s about the ascogonium,  which i s d i f f e r e n t i a t e d i n t o a basal c e l l , an ascogonium and a trichogyne which i s continuous with the ascogonium.  The trichogyne fuses with the  antheridium and the single male nucleus migrates and fuses with the female nucleus.  - 6 Olson i s o l a t e d and studied ten s t r a i n s of Ceratostomella fimbriata from l e s i o n s on sweet potato.  He found that they d i f f e r e d i n  pathogenicity to t h e i r suscept, i n morphology and c o m p a t i b i l i t y . The ascospore progeny of four s e l f - f e r t i l e strains included not only the parental s e l f - f e r t i l e t h e i r respective s e l f - s t e r i l e  (perithecial) strains but also  (non-parental) s t r a i n s .  Single conidium  i s o l a t e s which he made from monospore cultures were of the parental type.  Olson also found that long-necked p e r i t h e c i a developed on the  l i n e between s e l f - f e r t i l e some of the l a t t e r .  and s e l f - s t e r i l e  strains as well as between  It was found that i s o l a t e s from another s t r a i n  produced no p e r i t h e c i a either alone or i n combination with other s e l f sterile strains.  The ascospore progeny of p e r i t h e c i a formed by combinations  of strains comprised e i t h e r the parental types alone, or^both parental and recombination types,  suggesting that p e r i t h e c i a l production i s the  r e s u l t of at l e a s t two independently i n h e r i t e d genes.  Ascospores from  a homothallic s t r a i n gave r i s e to h e t e r o t h a l l i c s t r a i n s . The addition of 5JJ g thiamine t o Czapek's medium was e s s e n t i a l for p e r i t h e c i a l production by i s o l a t e s which were s e l f - f e r t i l e dextrose agar.  on potato  However, the a d d i t i o n of t h i s growth substance to the  l a t t e r substratum d i d not induce the formation of p e r i t h e c i a i n n a t u r a l l y self-sterile  strains.  Olson also found that s e l f - f e r t i l e  cultures of Ceratostomella  fimbriata i s o l a t e d from Hevea rubber trees i n Mexico and sweet potato from the United States, were s i m i l a r morphologically but were not cross pathogenic.  Both s t r a i n s gave r i s e to s e l f - f e r t i l e  and  self-sterile  strains among single ascospore cultures i s o l a t e d from them. week-old cultures of s e l f - s t e r i l e  He spermatized  sweet potato strains with spores from  - 7 s e l f - f e r t i l e rubber cultures and found that p e r i t h e c i a developed i n three days.  Ascospores from these p e r i t h e c i a germinated very poorly.  Only two  cultures out of three hundred produced cultures which were s e l f - f e r t i l e . One was pathogenic to both sweet potato and rubber sucepts and one was pathogenic to rubber only.  Ascospore i s o l a t i o n s from the two cultures  produced s e l f - f e r t i l e and s e l f - s t e r i l e as the parent.  s t r a i n s with the same pathogenicity  Hybridization occurred i n the experiment between the strains  of Ceratostomella fimbriata i s o l a t e d from sweet potato and from rubber. Hensen and Snyder (16) reported that crosses involving male and female mutants of C. fimbriata and hermaphroditic types demonstrated independently inherited f a c t o r s f o r each sex.  The presence o f both r e -  sulted i n hemaphroditism. Pontis (17) made cross ^inoculation experiments using the pathogens of both the coffee and sweet potatoes.  He iiinoculated sweet potato  roots by dipping them i n a spore suspension of conidia, ascospores and mycelium from a culture of the coffee i s o l a t e d and incubated at room temperature.  At the same time coffee trees were  fimbriata from sweet potatoes.  iusoculated with C.  None of these cross .inoculations produced  i n f e c t i o n , i n d i c a t i n g that the two diseases are caused by d i f f e r e n t strains of the same fungus.  A MORPHOLOGICAL STUDY  The germination, growth and development of a single conidium was followed.  The conidium i n i t i a t e d germination on sweet potato medium  at room temperature (24°C - 26°C) four and a h a l f hours a f t e r  isolation.  By the sixth hour the germ tube had attained a length equal to that of the conidium.  Ten hours a f t e r i s o l a t i o n the f i r s t septum was observed  and two hours l a t e r conidia formation started, apparently by fragmentation of the mycelium.  These conidia contained abundant o i l droplets.  These  spores germinated as d i d the parent conidium and i n t h i r t y - s i x hours the f i r s t impressions of a hyphial colony became apparent.  Asexual Reproduction Ophiostoma fimbriatum produces two types of asexual  spores.  One type i s the thin-walled hyalinegenerally c y l i n d r i c conidium and the other i s the heavy-walled, olive-brown oval or pear-shaped chlamydospore. The conidium has been observed to be produced i n two ways.  On  the surface of the medium fragmentation of the terminal portion of the a e r i a l hyphae has been observed to occur along the septa of the c e l l s r e s u l t i n g i n a chain of conidia set end on end.  The length of the hyphal  c e l l s , and hence the length of the r e s u l t i n g conidia appeared to be dependent on the culture medium used f o r growth.  The second process of  conidia formation i s more commonly known and i s described i n some d e t a i l by Lehman (4).  This method of formation involved the endogenous production  of the spores which i s c h a r a c t e r i s t i c of Ceratostomella.  The spores were  produced from inside the terminal c e l l of the conidiophore.  The walls of  the new spore were generated from the protoplast which was f i r m and entire  - 9 by the time the conidium was pushed out from the t e r m i n a l opening of the conidiophore.  The formation of the endoconidial w a l l was obviously not  the r e s u l t of the l o n g i t u d i n a l s p l i t t i n g of the conidiophore w a l l as claimed by B r i e r l e y (18) f o r the endoconidia of T h i e l a v i a b a s i c o l a (B & B r . ) . The w a l l s of the new spores appeared t o be generated by the c e l l p r o t o p l a s t . There appeared t o be two f a i r l y d i s t i n c t types o f conidiophores.  One  type was morphologically i n d i s t i n g u i s h a b l e from the t y p i c a l hypha of the fungus.  The other appeared to be more h i g h l y s p e c i a l i z e d f o r the production  of endoconidia.  The base of the c e l l was r e l a t i v e l y l a r g e and tapered  g r a d u a l l y to the t i p .  These c e l l s were longer and the basal diameter wider  than the producing c e l l s of the p r e v i o u s l y described conidiophore.  The  l a t t e r type of conidiophore appeared t o be a more p r o l i f i c producer of conidia.  These hyaline conidia were capable of germinating as soon as they  were produced from w i t h i n the sporophore. The other was a heavy-walled dark o l i v e spore produced endogenously from w i t h i n the s p e c i a l i z e d mycelium and a l s o by a process of budding. The endogenous manner of production of t h i s spore has been questioned by Lehman who contended that the protoplast was distended from the ruptured t i p before the conidium was a b s t r i c t e d .  These spores apparently contained  abundant food reserves and were observed t o be produced not only t e r m i n a l l y at the end of the conidiophore, but a l s o i n i n t e r c a l a r y p o s i t i o n s along hyphal strands.  As a r e s u l t of these observations, these spores are r e -  ferred t o throughout t h i s paper as chlamydospores.  The f i r s t spore  formed i n a chain was observed t o possess two d i s t i n c t w a l l s . Harter and Weimer (19) reported t h a t these spores germinated rarely.  Under the conditions of study none of these spores have been  - 10 -  observed to germinate to produce a germ tube or mycelium.  On several  occasions, however, the chlomydospores d i d produce other chlomydospores by budding ( P l a t e d  ).  There has been no reference i n the l i t e r a t u r e  reviewed i n d i c a t i n g t h i s type of spore formation i n Ceratostomella.  The  end r e s u l t was a number of spores eet end on end s i m i l a r to those produced i n an endogenous manner.  The mature pear-shaped spores were about 15/± to  1 9 y long and 9jJ- to 11 y. wide. about 6jj. i n diameter.  The mycelia producing these spores were  In no case -was; the formation of chlomydospores  observed on the surface of the medium.  A l l chlomydospore formation  appeared t o be isolated i n the medium. Both types of the asexual spores were one-celled and uninucleate. . Development of the Perithecia On the seventh day a f t e r germination, minute globular masses were seen scattered among the mycelium i n the colony.  On examination  under the binocular microscope i t was found that these masses consisted of an agglomeration of a few intertwined hyphal strands and a minute drop of colorless l i q u i d . disintegrated.  When touched with the point of a needle the mass  The development of one of these bodies was observed and  i t was found that the mass gradually became darker i n color from opaque to ochre to dark brown.  The mass became increasingly compact and the  mycelium appeared to branch profusely.  By the t h i r d day an almost black,  f i r m globular body was quite apparent under the binocular microscope. The beak was the l a s t part of the f r u i t i n g body t o be formed.  By the end  of the fourth day the black globose and beaked f r u i t i n g body was completely formed.  The beak was fimbriate.  On the f i f t h day a gelatinous mass of  - 11 -  spores accumulated at the fimbriate t i p of the f r u i t i n g body. Microscopic examination of sections of the f r u i t i n g body at various stages of i t s development f a i l e d , at f i r s t , t o give evidence of asci.  This was due to the deliquescent nature of the ascus walls as  described by E l l i o t t  (5).  It i s assumed that a s c i and p e r i t h e c i a l develop-  ment occur simultaneously and that the a s c i attained maturity and d i s integrated before the perithecium was f u l l y formed leaving a mass of free ascospores i n the p e r i t h e c i a l c a v i t y .  The gelatinous matrix i n which the  ascospores were exuded probably originated from the disintegrated a s c i walls and from the i n t e r n a l w a l l of the perithecium.  The one-celled  ascoperes occurred In hundreds i n these gelatinous exudations at the t i p of the p e r i t h e c i a .  These masses were whitish-opaque at f i r s t but about  fourteen days l a t e r they turned a smoky-yellow c o l o r .  At t h i s stage,  ascospores, while s t i l l attached to the t i p of the p e r i t h e c i a , germinated producing slender hyphae which produced hyaline conidia as described before. The globose portion of the;perithecia had a diameter of lCO)i 120JUL.  to  The length of the beak ranges from 180y to 2Q0y. The ascospores  are helmet-shaped and about 2js i n diameter (Plate I i ) . Another attempt at sectioning the p e r i t h e c i a proved more successful. The p e r i t h e c i a were picked out from the cultures, fixed and k i l l e d i n an a l c o h o l / a c e t i c acid mixture f o r 20 minutes and then d i r e c t l y i n f i l t r a t e d and imbedded i n p a r a f f i n wax.  Hand sections were made and by t h i s method,  two i n t a c t a s c i containing ascospores i n the p e r i t h e c i a l c a v i t y were obtained i n separate sections.  The a s c i were i r r e g u l a r l y sack-shaped and  while the number of ascospores could not be c l e a r l y d i s t i n g u i s h e d , four  - 12 seemed to be apparent i n each case. In cultures 12 days o l d a d i s t i n c t acetate-like odor of some apparently organic substance was d i s c e r n i b l e .  The odor increased i n  i n t e n s i t y u n t i l the colony stopped growth about three weeks l a t e r when the margin of the plate was reached.  - 13 A CYTOLQGICAL STUDY  The object of t h i s study i s to observe the nuclear structure and mode of d i v i s i o n i n the r e s t i n g and germinating conidia of the fungus. Although the germination of a spore appears to be a simple physical phenomenon, there are many small but e s s e n t i a l l y  constant  differences i n the germination of various fungus spores.  cytoplasmic I t i s understood,  for example, that at spore germination the nucleus divides and enters the germ tube, moves towards the t i p , divides again, one h a l f going to the growing point of the tube and the other h a l f remaining behind.  It  is  also known that the time at which the f i r s t nuclear d i v i s i o n occurs may vary.  Some n u c l e i divide before the apparent i n i t i a t i o n of the germ tube  while others divide l a t e r i n i t s  development.  The exact mechanism of the d i v i s i o n of the nucleus i n many fungi i s dbill an open question.  Some workers state that the d i v i s i o n i s b a s i c a l l y  the same as that which occurs i n the c e l l s of the higher p l a n t s , involving the accepted theories of mitosis and meiosis. (20,  Robinow and other workers  2L) are of the opinion that the n u c l e i of some of these lower forms  do not undergo any organized d i v i s i o n but divide by a more or l e s s random p u l l i n g apart of chromatin material which r e s u l t s i n two n u c l e i .  Materials and Methods Using the method described on page 40 f o r studies on the induction of mutant s t r a i n s , conidia were prepared f o r staining after growth had been i n i t i a t e d on d i a l y z i n g membrane.  It was found that a f t e r four and a h a l f  hours a t 27°C on potato dextrose agar, germ-tube i n i t i a l s were apparent on the majority of spores and by the f i f t h hour germ-tube elongation was well on i t s way.  By f i x i n g conidia on the membranes at i n t e r v a l s during  - 14 the t h i r d to sixth hour a f t e r planting, a suitable range of various stages of germination was  obtained.  The f i x a t i v e used was made up of 3 parts 95$ ethyl alcohol and 1 part g l a c i a l a c e t i c a c i d . into the f i x a t i v e . 10 minutes.  The membrane squares were placed d i r e c t l y  The material was k i l l e d and f i x e d f o r a period of  Throughout t h i s staining period small stenders e s p e c i a l l y  constructed f o r staining procedures using microscopic cover s l i p s were used.  The membranes were then placed i n 10% ethyl alcohol f o r 10  minutes and then hydrolyzed i n IN hydrochloric acid at 60°C f o r 10 to 13 minutes.  A f t e r washing i n d i s t i l l e d water f o r about 10 minutes, the  membranes were stained f o r 1 hour i n buffered Giemsa (buffered at pH 6.2).  The membranes were washed i n tap water before mounting i n water  f o r immediate observation.  Observations The r e s t i n g conidium had an average length of 16>1 average width of 6jJL.  and an  The conidium usually contained two large o i l  droplets at opposite ends and abundant cytoplasmic material containing many highly r e f r a c t i v e bodies. nucleus c e n t r a l l y located.  The conidia were uninucleate with the  The r e s t i n g nucleus stained blue as a dark  crescent-shaped homogenous mass with a d e f i n i t e l i g h t spherical area i n the arms of the crescent which was interpreted to represent the central body. Immediately preceeding the appearance of the germ-tube i n i t i a l s , the condensed homogenous nuclear mass gradually assumed a randomly organized r e t i c u l a t e system of chromatin material. could be i d e n t i f i e d .  Definite strands  The exact number, however, could not be d e f i n i t e l y  - 15 ascertained because of the r e t i c u l a t e nature of the mass.  Seemingly the  number of these strands vary between three and s i x , appearing as i r r e g u l a r l i n e a r bodies i n diverse shapes of Vs. The germ-tube i n i t i a l appeared about four to f i v e hours a f t e r planting.  More than one apparent germ-tube i n i t i a l was observed on conidia  but i n no case d i d more than one germ-tube develop.  The germ-tube i n i t i a l  r a r e l y appeared c e n t r a l l y , opposite to the nucleus.  In most cases the  i n i t i a l s arose nearer to one pole of the conidium, evidently along a thinner area along t h i s part of the c e l l w a l l . The f i r s t nuclear d i v i s i o n was never observed to occur before the germ tubes attained a length of about ky> .  The nuclear apparatus of  chromatin strands moved from i t s central p o s i t i o n i n the conidium towards the area of the germ tube i n i t i a l .  At t h i s s i t e there appeared to be a  mechanical separation of the diffuse r e t i c u l a t e material with the formation of a narrow isthmus across the point at which the germ tube leaves the conidium.  After the d i v i s i o n , one-half of the material migrated to the  t i p of the germ tube and the remaining h a l f moved back to i t s o r i g i n a l central l o c a t i o n i n the conidium.  The entire structure i s f i l l e d with  cytoplasmic material. The second nuclear d i v i s i o n was unique since i t was not the nucleus at the t i p which divided but, by a process s i m i l a r t o the  first  nuclear d i v i s i o n , the parent r e t i c u l a t e nucleus again moved toward the point of germ tube i n i t i a t i o n and divided as before.  One daughter nucleus  returned to the o r i g i n a l p o s i t i o n , while the other took i t s behind the nucleus at the t i p .  Thus f a r the germ tube was  containing two n u c l e i and no cross w a l l s .  position coenocytic,  - 16 At about the time of the t h i r d nuclear d i v i s i o n , a c e l l wall was observed between the two first-formed n u c l e i i n the germ tube.  Five  such d i v i s i o n s , involving the parent nucleus i n the formation of new nuclei f o r the elongating germ tube, were noted before a d i v i s i o n of the nucleus at the t i p of the germ tube was observed.!Plate  III).  Discussion No d e f i n i t e mitotic figures were observed i n any of the nuclear divisions.  The organization of chromatin from nuclear material appeared  to be s i m i l a r to that which occurs i n the prophase i n mitosis i n the c e l l s of higher plants.  The formation of new nuclei seemed t o be a random  p u l l i n g apart of the chromatin material which resulted f i n a l l y inf •> . two nuclei.  The l i g h t area interpreted as representing a central body was  seen only i n the resting n u c l e i and was not observed at the time of the organization of the nucleus into chromatin m a t e r i a l , nor during d i v i s i o n . Various techniques involving the use of methyl alcohol and formalin for the f i x a t i o n and k i l l i n g processes resulted i n great  distor-  t i o n of the spores and induced the formation of a r t i f a c t s i n the cytoplasm. The ethyl alcohol and g l a c i a l a c e t i c a c i d mixture which was f i n a l l y used appeared to be a milder f i x a t i v e and caused l i t t l e  distortion.  It was  observed that the duration and temperatures at which hydrolysis was carried out were c r i t i c a l . It i s thought that the repeated parental nuclear d i v i s i o n s might have been due to energy stored up i n the conidia and that the  capacity  for such d i v i s i o n s was retained u n t i l the new c e l l could obtain those nutrients which allow the d i v i s i o n of the new nucleus at the t i p of the germ tube to occur.  - 17  -  THE EFFECT OF VARIOUS CONCENTRATIONS OF THIAMINE HYDROCHLORIDE ON THE GROWTH AND  SPORULATION OF "STRAINS" OF  OPHIOSTQMA FIMBRIATUM Barnett and L i l l y (22) have shown that thiamine i s necessary for the production of p e r i t h e c i a i n the various species of Ophiostoma and Ceratostomella.  Beaxlle and Tat urn (23) found that some mutants of  Neurospora were unable to 'grow i n c e r t a i n nutrient media which lacked a vitamin or amino a c i d which the 'wild* type was able t o synthesize but which the mutant was unable to produce.  F r i e s (24) found that mutants of  Ophiostoma multiannulatum were unable to sporulate because they lacked the a b i l i t y to synthesize thiamine or other chemicals necessary f o r growth and sporulation.  Methods and Materials In t h i s experiment a modified Czapel Bacto Agar was used as the basal medium.  The various concentrations of thiamine hydrochloride used,  are shown i n table 1.  Two  'strains  1  (4x and 2x) and the o r i g i n a l 'wild  Table 1 The E f f e c t of Thiamine Hydrochloride  on  Growth of Ophiostoma fimbriatum (Age of Cultures 38 days at 27°C)  T  .  .  ThHCl gms/LOOO ml Growth i n cm. medium CfB  (Average of 4 plates/treatment) : : 4x 2x  1  0.5  4.3  5.0  4.3  2  1.0  4.3  5.9  3.8  3  1.5  4.3  5.9  4.0  4  2.0  4.5  6.0  4.0  - 18 -  type  1  CfB were grown under s i m i l a r treatments and the average colony  diameter i n four r e p l i c a t e s was used as the growth c r i t e r i o n .  Observations Isolate B.  In treatment 1 some zonation was present i n the  cultures but t h i s was not very d i s t i n c t .  The mycelia were very i r r e g u l a r  i n dimensions and d i r e c t i o n of growth, feeble i n appearance and septation and c e l l size were v a r i a b l e .  No chlamydospores were present but conidia  were p l e n t i f u l on the surface.  Neither perithecia nor ascospores were  observed. In treatment 2 the cultures were s i m i l a r t o those i n the f i r s t treatment except that under these conditions chlamydospores were produced and conidia were more abundant. The cultures i n treatment 3 were s i m i l a r t o those i n treatment 2.  With the maximum amount o f thiamine hydrochloride, (50 mgm. Th HCl/25  cc. p l a t e ) the mycelium was s t i l l irce;gular and feeble. contained more o i l droplets than i n the f i r s t treatment.  The c e l l s , however, Chlamydospores  were present but they were few and r e l a t i v e l y small. Isolate 4x.  With the lowest concentration of thiamine hydrochloride,  mycelial growth was very sparse and the mycelium was very f r a g i l e , forming chains of conidia of i r r e g u l a r s i z e s . periphery of the colony* i n c e l l s of various s i z e s .  This occurred e s p e c i a l l y on the  Septation of the mycelium was variable, r e s u l t i n g The cultures were r i c h with p e r i t h e c i a l i n i t i a l s  but no mature p e r i t h e c i a were observed.  Pigmentation was sparse throughout  the cultures and chlamydospores were few.  The cultures i n the second  treatment contained many more chlamydospores which were budding i n several places.  The central areas were more highly pigmented than the cultures i n  - 19 the previous treatment.  This area a l s o contained more p e r i t h e c i a l i n i t i a l s .  The cultures were more heavily pigmented and the mycelial septation was very inconstant.  The c e l l s varied i n size from  J>yx  to IQyx i n length.  Isolate 2x. The cultures of the f i r s t treatment contained no Surface mycelium but surface conidia were p l e n t i f u l . i n the medium but was very sparse.  Mycelium was present  No chlamydospores were observed and  the cultures appeared to be completely without pigment.  In the  second  treatment cultures were similar to those described above except thqt c o n i d i a , which were produced i n an endogenous manner, were observed i n the medium. In treatment 3 there was only a f a i n t pigmentation toward the centre and abundant surface conidia were formed, apparently by fragmentation of the a e r i a l mycelium. Under the conditions of t h i s experiment the various concentrations of thiamine hydrochloride appeared to affect only the production of chlamydospores and o i l droplets i n the mycelium i n the cultures of Isolate CfB.  Pigmentation appeared to s t a r t c e n t r a l l y i n the mycelium which was  submerged i n the medium i n the cultures of Isolate 4x. The a e r i a l mycelium was l i g h t colored.  The chia>mydospores were produced i n the medium  and were dark olive-green i n c o l o r .  - 20 -  THE EFFECT OF COLCHICINE ON THE GROWTH AND SPORULATION OF STRAINS* OF OPHIOSTOMA FIMBRIATUM f  The use of colchicine f o r the production of p o l y p l o i d mutants i n plants has attained widespread acclaim, e s p e c i a l l y i n the f i e l d of plant breeding and genetics.  The effect of colchicine appears to be  closely associated with the spindle f i b r e s .  E i g s t i and Dustin (25) are  of the opinion that the action of the drug involves the whole c e l l . Colchicine i s known to block the mechanism that r e g u l a r l y c a r r i e s the chromosomes to t h e i r respective poles. substrate where the a l k a l o i d a c t s .  Hence the spindle f i b r e i s the  Dustin (26) thinks that the basic  course for a mitotic arrest undoubtedly i s to be found i n the chemistry and physiology of the spindle f i b r e and attending mechanisms. workers (Ostagren G . (27),  and E i g s t i and Dustin (25))  Some  consider that the  fundamental r e l a t i o n s h i p between the drug and the spindle f i b r e i s a quantitative intermolecular r e a c t i o n , since the concentration of the drug i s a c r i t i c a l factor i n i t s capacity to a l t e r the morphological character and nuclear apparatus of the c e l l s .  Levan (28),  among many other workers,  has successfully demonstrated the remarkable a c t i o n of the drug.  Treated  c e l l s have the capacity to recover and segregate a b i p o l a r spindle mechanism for the promulgation of normal mitosis again. Gaulden and Carlson (29) and Wade (30) have shown that gene changes and chromosome repatterning do not occur i n c e l l s treated with the drug. found.  Changes comparable to those produced by X-rays have not been  Only mutation involving the doubling of chromosomes have been  known to occur. true mutagen.  As a r e s u l t i t i s incorrect to c l a s s i f y the drug as a From a review of the l i t e r a t u r e the a c t i o n of colchicine  - 21 on the fungi appears to be very v a r i a b l e .  Vanderwalle (31) found that the  drug hindered the production of conidia i n Diaporthe p e r n i c i o s a .  Blakeslee  (32), working with several species of f u n g i , d i d not observe any change due to the action of the a l k a l o i d .  Several workers (Levan (33)» Beams (34),  Laur (35), Vandendries (36)) f a i l e d to produce any changes i n cultures of Saxharomyces cerevisieae.  Sinto and Yuasa (37) were able to produce  c y t o l o g i c a l changes i n t h i s yeast while E i g s t i andMs associates obtained enlarged c e l l s by colchicine treatment.  Grace (38) observed i n h i b i t i o n  while Richards (39) observed stimulation i n the production of yeast  cells.  E i g s t i and Dustin stated that changes i n the s i z e s of c e l l s within a culture and d i r e c t a c t i o n upon the growing organisms indicate that the drug has some influence upon the growth processes related to increase i n size.  These changes are not transmitted t o succeeding  generations.  Gorter (40) has shown that colchicine modifies the c e l l wall structure during the process of formation i n fungi and algae.  The most widespread  concentration of colchicine used i n experimental plant studies appears to be a 0.2$ s o l u t i o n .  Hindmarsh (41) used a 0.1$ solution to prevent forma-  t i o n and for the destruction of the spindle mechanisms .in; onion root t i p cells.  Nishiyama (42) used a 0.05$ solution to produce pentaploid hybrids  i n oats.  Walzel (43) used a 0.01$ solution to show i n h i b i t i o n of  seedling  elongation, while Alcaraz and his associates (44) used 0.2 and 0.5$ solutions to produce t e t r a p l o i d tobacco p l a n t s .  Mader (45) obtained  c e l l u l a r abnormalities i n Marchantia spore germination i n 0.2 - 0.4$ solutions.  Most of the experimental work on fungi was done i n concentrations  of c o l c h i c i n e , ranging from 0.01$ to 2$.  - 22 Materials and Methods In a l l treatments potato dextrose agar was used as the basal medium.  Five ninety m i l l i l i t r e portions of medium were s t e r i l i z e d by  a u t o d a v i n g i n the usual manner.  At the same time f i v e test tubes, each  containing 10 ml. d i s t i l l e d water and 0.5 gnu, 1.0 gm., 1.5 gm. and 2.0 gm. colchicine respectively (the 5th tube was used as a control) were sterilized.  When the medium was cooled to about 50°C the various con-  centrations of colchicine were added to the f l a s k s of medium to make four treatments containing 0.5$, 1.0$, 1.5$ and 2$ c o l c h i c i n e . the f i f t h f l a s k was used as a c o n t r o l .  The medium i n  The flasks were then s t i r r e d to  ensure the proper mixing of the medium with the added colchicine s o l u t i o n . From each f l a s k four plates were poured. each s t r a i n used i n the experiment. centrally.  This process was repeated f o r  On each plate one planting was made  The plates were incubated at 27°C.  Observations were made  from the tenth to f i f t e e n t h day a f t e r p l a n t i n g .  Observations Table 2 The E f f e c t of Colchicine on the Growth of Ophiostoma fimbriatum (Cultures 16 days o l d , incubated at 27°C)  Treatment  Colchicine gms/l00 ml. medium  Diameter of Growth i n cm. (Average of 4 plates/treatmant) Cf.B  4x  2x  1  0.5  4.4  5.6  3.5  2  1.0  4.4  6.0  3.5  3  1.5  4.6  7.0  4.3  4  2.0  4.5  5.5  4.5  5  control  5.0  8.0  5.9  - 23 Isolate C f . B .  In treatment 1 and 2 the size and the  of the mycelium were very i r r e g u l a r . many cases collapsed.  septation  The mycelium was i n d i s t i n c t and i n  No chlamydospores were observed i n these cultures  but there were abundant conidia on the surface of the medium. and banding i n a l l cultures were prominent.  Zonation  In treatment 3 the zonation  was s t i l l manifest and the mycelium was i r r e g u l a r as i n the f i r s t  treatment.  In these cultures chlamydospores were present but they were comparatively very few.  The hyaline conidia on the surface were p l e n t i f u l .  Many dramatic  abnormalities involving the general morphology, germination and budding of these spores were observed.  In some cases the surface vegetative  mycelium produced l a t e r a l pear-shaped protuberances. germinated to produce s i m i l a r i r r e g u l a r 'buds'.  Numerous conidia  These bodies varied  from pear-shaped to club-shaped structures and many were detached and strewn on the surface of the culture. dumb-bell shaped.  Some conidia were found to be  These structures were a l l thin-walled and hyaline.  (Plate 4) More chlamydospores were present i n treatment 4 than any other treatment.  The zonation i n the cultures was s t i l l marked, the mycelium  was s t i l l weak and i r r e g u l a r , containing abundant o i l droplets.  The  surface conidia were i n copious amounts with l i b e r a l budding throughout the cultures. Isolate 4x. were s i m i l a r .  Cultures of t h i s i s o l a t e i n treatments 1 and 2  Large quantities of conidia were formed by fragmentation  of the f r a g i l e surface mycelium.  The conidia were generally i r r e g u l a r .  Chiamydospores were very numerous and were produced i n terminal i n t e r c a l a r y and l a t e r a l positions on the vegetative mycelium (Plate IV). Cultures i n treatments 3 and 4 were s i m i l a r and noteworthy by  - 24 the f a c t that none of these produced chlomydospores. i n large quantities i n various sizes and shapes.  Conidia were produced  Some were small and  almost s p h e r i c a l , others were much more elongated and c y l i n d r i c a l i n form. Isolate 2x. chlomydospores.  Cultures i n the f i r s t two treatments d i d not produce  Conidia, o r i g i n a t i n g from inside the mycelium (endoconidia),  were formed i n f a i r abundance i n the medium.  A e r i a l mycelium was absent,  since apparently a l l had been u t i l i z e d i n the production of  surface  conidia. The cultures i n treatments 3 and 4 contained almost no pigment i n contrast to the cultures i n the previous treatments.  At the higher  concentration of colchicine there was no evidence of pigmentation.  Surface  conidia were widespread but no conidia were observed to be produced i n the medium.  Chlomydospores were not produced i n these c u l t u r e s .  The only apparent difference between the zones of banding i n the C f . B i s o l a t e was the r e l a t i v e abundance of mycelium.  These cultures  demonstrated c e r t a i n p e c u l i a r i t i e s i n the morphology and germination of conidia.  The cultures of i s o l a t e 4x were most p r o l i f i c i n growth.  These  c u l t u r e s , apart from portraying c o n i d i a l abnormalities, also exhibited idiosyncrasies i n the formation of ehlomydospore. produce chlomydospores i n any treatment.  Isolate 2x did not  The colchicine appeared to have  affected the pigment system of t h i s s t r a i n i n some way. It i s to be noted i n p a r t i c u l a r that neither p e r i t h e c i a nor ascospores were borne i n any c u l t u r e . The control cultures of Isolate B produced both types of asexual spores and p e r i t h e c i a y i e l d i n g ascospores a l l i n the t y p i c a l manner. Pigmentation was normal, as described i n the morphological study.  Isolate  - 25 -  2x remained true to form i n the control cultures.  This i s o l a t e produced  abundant conidia but yielded neither chlomydospores nor p e r i t h e c i a with ascospores.  The control cultures of Isolate 4x were s i m i l a r to those  described i n the o r i g i n a l  isolations.  Apart from the morphological changes and peculiar sporulation c h a r a c t e r i s t i c s i n the cultures, the growth rate of a l l i s o l a t e s i n the control cultures were greater than the same i s o l a t e s with colchicine incorporated i n the basal medium.  Undoubtedly the drug affects the  physiology of growth as well as of reproduction.  - 26 -  THE EFFECT OF CAMPHOR ON THE GROWTH AND SPORULATION OF OPHIOSTOMA FIMBRIATUM  Camphor has been used i n a wide v a r i e t y of plants to induce morphological and c y t o l o g i c a l v a r i a t i o n s .  The a c t i o n of camphor on plant  c e l l s appeals to be a complex one, f o r Deysson (46) i n France obtained very diverse r e s u l t s , using d i f f e r e n t concentrations of the chemical on onion root t i p s .  At a concentration of 0.02$ to 0.03$ there were no  abnormalities i n c e l l size but there were c y t o l o g i c a l aberrations.  At  much lower concentrations (0.015$ to 0.012$) such enlargements were induced i n the area of c e l l elongation.  Won (47) developed 'giant*  cells  i n bacteria by incorporating from 25 mg. to 55 mg. of camphor i n 100 ml. blood agar.  It i s his opinion that the r a p i d penetration of camphor i n t o  the b a c t e r i a l c e l l was an important factor i n the production of veriants.  'giant'  Subramanian and Roa (48) have indicated from t h e i r experiments  that camphor had induced gene mutations i n several d i r e c t i o n s i n yeasts. Kostoff (49) found that a saturated s o l u t i o n of camphor i n h i b i t e d the growth of mycelium of P e n i c i l l i u m sp. growing on honey agar.  This  treatment apparently encouraged branching, some of the branches being thicker and occasionally forming whole chains of giant c o n i d i a .  Camphor  i n large doses over a prolonged period prevented the formation of c o n i d i a . He noted that the giant c o n i d i a , when i s o l a t e d , germinated and maintained the production of giant conidia through several t r a n s f e r s .  Kostoff thinks  that the gigantism was probably due to the doubling of the chromatin material.  - 27 Materials and Methods Comparatively high concentrations of camphor were used i n these experiments to ascertain i t s effect on the test organisms. D i f f i c u l t y was encountered i n d i s s o l v i n g more than 0.2 gms. of camphor i n s t e r i l e water, even by autoclaving. The camphor was dissolved and autoclaved separately and l a t e r added to double strength potato dextrose agar. Medium containing 0.2$ and camphor constituted treatment 1 and 2, respectively.  0.4$  Treatment 4 was made up of  plates containing no camphor and was employed as the c o n t r o l . Four plates with a c e n t r a l planting of Isolate C f . B were used i n each treatment. Observations Table 3 The E f f e c t of Camphor on the Growth and Sporulation of Ophiostoma fimbriatum ( A l l cultures were incubated f o r 22 days at 2 7 ° C . Treatment Camphor Diameter Sporulation gms/100 m l . of growth Ascospores A e r i a l EndoChlamydospores medium i n cm.* conidia conidia 1  0.2  6.4  X  XXX  XXX  X  2  0.4  7.0  XXX  XXX  XXX  XX  3  control  5.4  X  XXX  X  XXXX  The control cultures of the organism produced both perithecia and ascospores but only i n r e l a t i v e l y small numbers. The perithecia were found to be aggregated around the central core and appeared s p o r a d i c a l l y , and much l e s s extensively, on the rest of the colony. The production of  - 28 -  chlomydospores was very extensive and the a e r i a l conidia were numerous. The cultures i n Treatment 1 were darker olive-green i n appearance when compared with the control c u l t u r e s .  The y i e l d of p e r i t h e c i a and ascospores  were accelerated i n these c u l t u r e s .  Copious amounts of conidia were  produced i n the medium i n an endogenous manner.  The hyaline c o n i d i a ,  both a e r i a l and submerged, were present i n great abundance.  There was  a marked reduction i n the amount of chlomydospores present.  The mycelium  was vigorous and regular containing numerous o i l globules.  The cultures  i n Treatment 2 were i n many ways s i m i l a r to those i n the previous treatment, bearing regular, vigorous mycelium. spores was greatly increased. the surface of the medium.  The production of pigment and sexual  Perithecia were evenly d i s t r i b u t e d throughout  Both the a e r i a l and endoconidia were p l e n t i f u l  and although there were much l e s s chlomydospores than i n the control plates these cultures contained more of these spores than those growing on the medium containing l e s s camphor.  Discussion No apparent morphological mutation occurred i n the cultures i n the above experiment.  It was noted, however, that camphor did affect  the growth and sporulation of the fungus markedly.  The rate of growth  was accelerated by the a d d i t i o n of camphor to the medium and an increasing amount of camphor appears to increase the vigour and growth of the  cultures.  D i f f i c u l t y was encountered i n d i s s o l v i n g more than 0.2 gms. of the camphor i n the medium even by autoclaving at 2 1 5 ° and 15 l b . pressure. Camphor seemed to bring about a greater production of p e r i t h e c i a and ascospores and a smaller amount of chlomydospores.  The control plates  contained more chlomydospores than e i t h e r of the camphor p l a t e s .  The  hyaline conidia were abundant i n a l l cultures. type of spore most e a s i l y formed by t h i s fungus. spore i n the medium was r e l a t i v e l y r a r e .  These appeared to be the The formation of t h i s  However, i n both camphor  treatments t h e i r formation i n the medium from inside the mycelium (endoconidia) was f a i r l y abundant.  These spores, which are more commonly  formed on the surface of the c u l t u r e , were amply produced but the method of production appeared not to be from inside the mycelium but by f r a g mentation of the a e r i a l mycelium.  - 30 -  THE EFFECT OF VARIOUS SOURCES OF NITROGEN ON THE GROWTH AND SPORULATION OF OPHIOSTOMA FIMBRIATUM  Barnett and L i l l y  (50) have demonstrated tha importance of  nitrogen i n the physiology of sporulation i n many f u n g i .  In t h i s  ex-  periment four different sources of nitrogen, including organic and i n organic substances,  have been used i n the basal medium.  Materials and Methods Asparagine and urea served as the organic sources while potassium n i t r a t e and ammonium sulphate were used as the inorganic sources of nitrogen.  These compounds constituted four treatments, the f i f t h treatment  being the c o n t r o l .  In each case an equivalent of 0.76 gm. of nitrogen  was employed i n 100 cc. of the basal medium.  This medium consisted of  the following materials: Malt Extract  10 gms.  Bacto-agar  10 gms.  D i s t i l l e d water  500 cc.  The nitrogenous compounds were a l l dissolved i n the  distilled  water before the a d d i t i o n of the other components of the medium. complete media were then s t e r i l i z e d by autoclaving.  The  Four plates were  formed from each treatment l o t and planted c e n t r a l l y with Isolate C f . B . The plates were incubated at 2 7 ° C .  Observations The growth of the cultures i n Treatment 1 was very vigorous with a uniform upper surface.  The underside appeared dark olive-green  - 31 Table 4  ..  The .eiffect Of Various Sources of Nitrogen on the Growth and Sporulation. of Ophiostonia fimbriatum (All  cultures grew on an equivalent of 0.76% nitrogen at 27^6 f o r 16 days)  Treatment Nitrogenous Amount Diameter Sporulation • compound (gm) of growth Ascospores A e r i a l EndoChlamydospores (cm)* conidia conidia  1  Asparagine  2  5.0  0  X  0  2  2.7  1.7  0  xxxx  X  0  1.9  0.6  0  XX  XX  XX  4  Potassium nitrate Ammonium sulphate Urea  0.8  —  5  Control  —  4.2  3  XXXX  no growth XX  XXXX  XX  XX  *Average of four plates with numerous dark-black spots indispersed throughout the c u l t u r e . The upper surface contained abundant a e r i a l mycelium and c o i n d i a . There was a widespread production of chlamydospores i n the cultures. The mycelium was heavily pigmented and was r e l a t i v e l y vigorous i n appearance. On closer examination, the dark spots seen from the underside of the plate proved to be aggregated clumps of densely pigmented mycelium. The growth mat was hard and leathery i n places. Neither perithecia nor ascospores were observed i n any cultures. Treatment 2 with potassium n i t r a t e produced c h a r a c t e r i s t i c changes i n the growth of the organism The colonies were very uniformly fawn-colored, both on the top side and underside The growth was very regular and the cultures had an even, mealg appearance. The mycelium was not as vigorous as the mycelium i n Treatment 1, and did not appear to be pigmented. The hyaline c y l i n d r i c conidia were produced i n copious  quantities  but^neither chlamydospores nor preithecia were  - 32 produced.  Table 4 shows that growth i n these plates was much l e s s than  i n the previous treatment.  In Treatment 3, ammonium sulphate was used  as the source of nitrogen.  The cultures here had a very l i m i t e d diameter  of growth as i s shown i n Table 4.  The undersides of the colonies were  extremely dark olive-green with an 0.5 mm. white periphery.  The upper  sides were l i g h t olive-green with a s i m i l a r peripheral area.  In general,  the colonies were r a i s e d and were of a leathery consistency.  Mycelial  growth was i r r e g u l a r and the mycelium was very f r a g i l e .  Conidia were  few and the chlamydospores, though present, were not p l e n t i f u l and a l l possessed r e l a t i v e l y t h i n walls.  No sexuality was observed i n the cultures.  The treatment which contained urea hindered a l l growth of the fungus. Because of t h i s f a c t and since the organism grew well on the control plates, i t was thought that e i t h e r the concentration of the urea was too high i n the medium or that s t e r i l i z a t i o n decomposed the urea and that the break-down products rendered the medium t o x i c to the fungal growth. To check these p o s s i b i l i t i e s , a number of plates were prepared containing various concentrations  of urea added t o the basal medium before and  a f t e r s t e r i l i z a t i o n when the medium had cooled t o 50°C, as shown i n Table 5.  The cultures were a l l very dark olive-green and almost black.  The  periphery of the cultures was i r r e g u l a r . The central areas (about 0.6 cm. diameter) were r a i s e d and l i g h t brown i n color.  They consisted of  weak, i r r e g u l a r , a e r i a l mycelium with abundant o i l droplets, conidia of variable shaped and r e l a t i v e l y few brown chlamydospores produced by budding.  The other parts of the colonies produced no chlamydospores and  only very few conidia. This experiment showed that urea was decomposed by heat  - 33 Table 5 The Effect of Concentration and Heat S t e r i l i z a t i o n of Urea on the Growth of Ophiostoma fimbriatum ( A l l cultures were grown at 27°C f o r 16 days)  Treatment  Amount of urea per 100 ml. basal medium (gm)  Diameter of Growth (cm) Average of 4 plates Urea Urea autoclaved not autoclaved  1  0.8  no growth  1.0  2  1.2  it  1.3  3  1.6  it  1.5  4  2.0  it  1.5  5  control  2.2  s t e r i l i z a t i o n into compounds that were t o x i c or compounds rendered t o x i c by the condition involved i n the study to the growth of the organism. The u n s t e r i l i z e d urea not only produced growth but the extent of the growth varied to some extent with the concentration of the urea i n the basal medium.  - 34 THE EFFECT OF COPPER ON THE GROWTH OF OPHIOSTOMA FIMBRIATUM  Mulder (51),  i n an a n a l y t i c a l study of s o i l s for micronutrients,  used Aspergillus niger as a microbiological assay f o r copper.  He found that  when a nutrient solution was p u r i f i e d from copper by charcoal treatment (shaking with amorphous charcoal) A s p e r g i l l u s developed only s t e r i l e mycelium.  white  The addition of ascending amounts of copper to the p u r i f i e d  medium induced sporulation with increasing pigmentation of the  spores  u n t i l f i n a l l y normal black conidia were produced at an upper concentration of 2.5  g i n 40 cc. of medium.  He concluded that copper had the apparent  function of an oxidation c a t a l y s t .  In a second s e r i e s of experiments the  copper impurity of the nutrient was removed by r e c r y s t a l l i z i n g the s a l t s . On a medium prepared from these s a l t s and provided with an adequate amount of copper, the growth of A s p e r g i l l u s was very poor.  He thought that the  nutrient medium p u r i f i e d with charcoal, though free from copper, contained some other substance which was removed i n the process of r e c r y s t a l l i z a t i o n of the nutrient s a l t s and the sugar. Since copper was shown to influence the formation of pigments i n A s p e r g i l l u s niger by Mulder (51) and Sakamura (52),  i t was decided  to t e s t the effect of copper-deficient nutrient on the growth of t h i s fungus (0. fimbriatum).  Methods and Materials The method of p u r i f i c a t i o n involved, i n a modified manner, the processes used by Mulder and Sakamura. prised the following ingredients:  The culture medium employed com-  - 35 Dextrose  20 gms.  Sodium n i t r a t e  2 gms.  Potassium dihydrogen phosphate  2 gms.  Sodium chloride  5 gms.  Magnesium sulphate D i s t i l l e d water Amorphous*  0.5 gms. 1000 ccs.  charcoal was shaken i n t e r m i t t e n t l y , over a period  of 30 minutes, with ample sulphuric a c i d .  The a c i d was f i l t e r e d out and  the charcoal residue was thoroughly washed with an excess of double d i s t i l l e d water.*  The residue was oven-dried at 6 0 ° C , f o r 3 hours.  This  charcoal was added to the l i q u i d medium i n excess (about 150 gms/500 c c ) . This mixture was shaken vigorously intermittently over a period of 2 hours, and then f i l t e r e d through f i l t e r paper, treated with d i l u t e sulphuric a c i d , washed with double d i s t i l l e d water and dried i n the oven. further p u r i f i c a t i o n calcium phosphate was used.  For  This phosphate was  f i r s t p u r i f i e d by shaking i n double d i s t i l l e d water (50 g m / l i t r e ) over a period of 4 hours during which the water was changed four times. mixture was f i l t e r e d as described before.  The  The p u r i f i e d calcium phosphate  was added to the culture solution (0.5 gm/lOO c c . ) and, using sodium hydroxide, the r e a c t i o n of the solution was adjusted to pH 5.5.  This  mixture was also shaken p e r i o d i c a l l y f o r 2 hours and f i l t e r e d as before. The glassware to be used was a l l f i l l e d with 1% solution of p u r i f i e d calcium phosphate and autoclaved f o r 20 minutes at 15 l b s . pressure.  The  solution was then poured out and the vessels washed with double d i s t i l l e d water.  Further precautions i n p u r i f i c a t i o n were not observed since i t  *  From glass s t i l l and r e c e i v e r .  - 36 -  was thought that these would be n u l l i f i e d by the traces of copper that may be present i n water and glass. 50 c c . of the p u r i f i e d medium were added to each of s i x 'copper free  1  250 cc. Erlenmeyer f l a s k s .  To another 300 cc. of p u r i f i e d medium  was added 30>Ag of copper from a solution of copper sulphate  (i.e.  Uj*g copper per 40 cc. media as compared to a minimum of 2.5 pg/hO cc. used by Mulder to produce normal black spores i n A s p e r g i l l u s ) .  This  medium was equally d i s t r i b u t e d i n t o s i x more f l a s k s as before.  A l l flasks  were planted with mycelial fragments from t h i r d generation cultures of Isolate B grown successively on ' p u r i f i e d medium'.  The inoculum con-  tained no spores and consisted e n t i r e l y of hyaline mycelium with a cottony appearance.  As a c o n t r o l , s i x f l a s k s containing 50 c c . of a  modified Czapek's solution were a l s o planted with the 'copper d e f i c i e n t ' inoculum. The eighteen flasks were placed on an automatic shaker at 5 p.m. and removed ayb 8.30 a.m. during the period of growth.  The cultures  were grown at room temperature and a t the end of 15 days, the growth i n each f l a s k was f i l t e r e d through i n d i v i d u a l l y weighed f i l t e r papers which were ovenj»dried i n desiccators at 3 0 ° C . f o r 12 hours.  The cultures on  the f i l t e r papers were then returned to the desiccators i n the oven and l e f t overnight before re-weighing.  Observations There were marked c u l t u r a l differences used.  i n the various media  The mycelium i n the cultures grown i n the Cu-deficient medium  contained no observable amount of pigmentation.  The mycelium was r e l a t i v e l y  - 37 very sparse and i r r e g u l a r i n c e l l size and i n the width of the hyphae. The cultures were completely s t e r i l e ,  neither sexual nor asexual  spores  being observed. The cultures grown i n the ' d e f i c i e n t medium', to which copper was added, showed marked pigmentation. of the culture was olive-green i n c o l o r .  The older mycelium i n the centre The mycelial t i p s were hyaline  and c y l i n d r i c a l conidia were endogenously produced.  The mycelium was  d e f i n i t e l y more vigorous i n appearance, with thicker walls and o i l i n most of the  globules  cells.  The cultures grown i n the modified Czapek's solution were darker olive-green i n appearance, i n d i c a t i n g a higher degree of pigmentation than that of the cultures growing i n the 'deficient medium' with copper added.  The mycelium was comparatively heavy-walled with regular d i s t i n c t  septation and although the hyphae t i p s were much l i g h t e r i n color than i n the older mycelium the presence of some degree of pigmentation i n these parts was apparent.  Most profuse growth took place i n t h i s medium,  and conidia and dark grey-green, pear-shaped chlamydospores were present. The hyaline conidia outnumbered the d a r k - c e l l e d chlamydospores by about 50:1.  The chlamydospores were produced mainly i n the older central area  of the c u l t u r e .  No mature p e r i t h e c i a were observed but numerous p e r i t h e c i a l  i n i t i a l s (as i d e n t i f i e d on cultures growing on agar) were observed i n the older mycelia.  Squash mounts of the i n i t i a l s at t h i s stage f a i l e d to  reveal any a s c i or ascospores.  The mycelium forming these i n i t i a l s wais  the darkest (brown-black) and contained more pigment than the other hyphae.  - 38 -  Table 6 The E f f e c t of Copper-Deficient Medium on the Growth of Ophiostoma fimbriatum  Weight i n grams of 10 day old cultures Culture No. Treatment 1 Cu-deficient Medium  Treatment 2 Deficient Medium • Cu  Treatment 3 Modified Czapek's Medium  1  0.050  0.10  0.21  2  0.045  0.10  0.25  3  0.050  0.09  0.20  4  0.042  0.12  0.23  5  0.047  0.11  0.20  6  0.051  0.09  0.24  0.045  0.102  Average  .22  Conclusions From the weights of the cultures grown i n the various media,  it  i s evident that copper deficiency affects the physiology of growth i n t h i s fungus.  A l l cultures grown i n the 'copper d e f i c i e n t '  l e s s growth than the other cultures.  medium produced  I t w i l l be noted that the average  weight of cultures grown i n Czapek's modified medium almost doubled the average of Treatment 1 and was also greater than that of Treatment 2. A discussion as to the probable reason f o r these d e f i c i e n c i e s has been treated e a r l i e r . It i s thought that the greater growth under Treatment 3 i s due  - 39 to the fact that the p u r i f i c a t i o n process i n Treatment 2 probably removed other necessary metals from the solution.  The medium under Treatment 3,  being unpurified, contained enough micronutrients f o r the metabolism of the organism used.  It i s also thought that apart from i t s effect on  pigmentation, copper appears to have some d i r e c t or i n d i r e c t effect on p e r i t h e c i a l formation.  F r i e s (53)  observed that a mutant s t r a i n of  0. multiannulatum. which could not synthesize i t s own tyrosine, remained without pigment.  From work with s t r a i n s of 0. fimbriatum i t was also found  that strains with l i t t l e  or no pigment (IX and 9X) produced no p e r i t h e c i a .  From these observations i t appears that copper and tyrosine  metabolism  may have some c o r r e l a t i o n with p e r i t h e c i a l formation. I t i s believed that p e r i t h e c i a l formation under Treatment 3 was i n h i b i t e d because of two probable reasons: 1.  Unsuitable  substrate  2.  Inadequate  aeration  Using a s i m i l a r medium with the a d d i t i o n of agar, t h i s fungus produced p e r i t h e c i a i n a 1 G day old c u l t u r e .  It i s thought,  therefore,  that the l i q u i d medium provided osmotic conditions which were unsuitable for p e r i t h e c i a l formation. A l s o , perithecia have been observed to occur s u p e r f i c i a l l y or only s l i g h t l y immersed i n the substrate whether the fungus i s growing on a r t i f i c i a l media or n a t u r a l l y on a susceptible host.  This tends to  indicate that aeration may play some role i n the formation of p e r i t h e c i a i n t h i s fungus.  Inadequate aeration i n the l i q u i d medium, i n spite of  intermittent shaking, i s probably one of the factors i n h i b i t i n g p e r i t h e c i a l formation i n Treatment 3.  - 40 -  From t h i s and other studies i t i s noted that hyaline conidia are the spores most e a s i l y formed i n culture followed by chlamydospores, and f i n a l l y by the sexual stage as n u t r i t i o n a l conditions improve.  - 41 THE INDUCTION OF MUTATIONS BY X-RAYS AND ULTRA-VIOLET RAYS  From the work of Timofeeff-Ressovsky  (54), Sinnott and h i s  colleagues (55) concluded that the frequency of mutation induced by X-rays i s d i r e c t l y proportional to the amount of r a d i a t i o n . They also found that X-rays of different wave-lengths produce equal numbers of mutations, provided that the amounts of the rays measured i n rontgens are a l i k e .  A l s o , a c e r t a i n amount of X-rays has  the same effect regardless of whether i t i s administered to the organism i n a short time from a powerful source or over a longer period from a weak source.  I f the same amount of rontgen units i s administered,  continuous treatments have the same effects as interrupted ones.  This  i s so since the mutagenic e f f e c t s of X - r a d i a t i o n are cumulative. Mutagenic changes brought about i n c e l l s treated with X - r a d i a t i o n are thought to be due to two types of nuclear aberrations, ( i ) genie, ( i i ) chromosomal. Zimmer and Delbruck (56) i n 1935 proposed the 'target theory* of induction of gene mutations by X-rays.  According to t h i s theory a  mutation i s caused by a single i o n i z a t i o n or atomic e x c i t a t i o n within a c e r t a i n 'target*.  It i s assumed that a single  target w i l l cause a gene change.  ' h i t * anywhere i n the  These changes are very l o c a l i z e d .  Chromosomal changes are usually more crude mechanical changes involving breakage of the chromosomes and r e s u l t i n g i n d u p l i c a t i o n s , translocations and i n v e r s i o n s .  deficiencies,  Non-disjunction and l o s s of  chromosomes are also frequent i n c e l l s treated with X - r a y s .  - 42 -  In contrast to gene mutations, the frequency of chromosomal changes induced by X-rays i s proportional roughly to the square of the dose administered as measured i n rontgens so that doubling the amount of treatment almost quadruples the production of chromosomal aberrations. The a c t i o n of Xerays may be interpreted as one producing breakages i n chromosomes, the number of breakages being proportional to the amount of treatment.  Sinnott and colleagues further state that a broken end of  a chromosome may r e j o i n other broken chromosomes and thus produce aberrations. I f , however, the end r e j o i n s the o r i g i n a l chromosome there w i l l be no change.  F i n a l l y , broken chromosomes may be l o s t . Muller (57) i n 1927 suggested that most of the effect of  i r r a d i a t i o n resulted from i o n i z a t i o n s within the chromosome i t s e l f . Wyss and his colleagues  (58) furthered t h i s view by inducing mutations i n  Staphylococcus aureus by chemical treatment of the substrate with hydrogen peroxide. The object of t h i s experiment i s to obtain mutant strains of t h i s fungus which d i f f e r from one another i n gross morphologic characteristics,  such as colony c o l o r , rate of growth, dimensions of the mycelium  and the production of sexual and asexual  spores.  Methods and Materials A l l induction treatments were done on spores or cultures of 0. fimbriatum obtained from Dr. W . J . Martin. Potato dextrose agar was used as the medium during treatment and for subsequent growth.  In the i r r a d i a t i o n of spores, several small squares  (about 1 cm. sq.) of d i a l y z i n g membrane which had been s t e r i l i z e d by b o i l i n g f o r 15 minutes i n a P e t r i d i s h , were placed c o n t r a l l y on a s t e r i l e  - 43 P e t r i dish containing about 25 cc. P . D . A . as shown i n Figure 1.  Using a  s t e r i l e wire loop, a suspension of spores -was; gently streaked over the membrane squares, care being taken to use a r e l a t i v e l y high d i l u t i o n of the spores.  The spores were i r r a d i a t e d immediately or allowed to germinate  and treated 6 to 8 hours a f t e r p l a n t i n g .  After treatment each membrane  square was transferred a s e p t i c a l l y and planted c e n t r a l l y on a s t e r i l e P . D . A . plate and the spores allowed to grow. For culture treatment, a (2 mm. diameter) d i s c of inoculum was planted c e n t r a l l y on a s t e r i l e plate and allowed to grow for 2 days at room temperature before being treated. In a l l cases treatments were done with the l i d of the P e t r i dish i n p o s i t i o n , corrections being made for the glass top and the distance between the surface of the organism and the l i d . Periodic observations of the plates were made f o r sectoring or abnormal growth and from such areas i s o l a t i o n s were made.  L a t e r , these  cultures were examined and compared with the o r i g i n a l culture f o r morphological difference. The machine used f o r i r r a d i a t i o n was a s u p e r f i c i a l X-ray u n i t . The plates were treated i n d i v i d u a l l y and were placed on a wax block to ensure the true dosage at a F . 5 . D . of 15.9 cm. 90 K . V . and 10 milliamperes constantly. aluminum f i l t e r was used.  In some treatments a g mm.  This e f f e c t i v e l y  but screened out soft r a d i a t i o n .  The machine was run at  reduced the rate of i r r a d i a t i o n  In some cases no f i l t e r was used.  From a review of the l i t e r a t u r e , iib appears that there i s a wide v a r i a t i o n i n the amount of r a d i a t i o n required to induce mutation even between closely related organisms.  - 44 Emmerling (59) treated the chromosomes of Zea mays with X - r a d i a t i o n and administered doses ranging from 400 r to 1000 r .  Deschuer and  Sparrow (60) i r r a d i a t e d T r i l l i u m erectum anthers with 50 r . delivered continuously for 35 - 45 seconds at a distance of 50 cm.  Fries  (53)  i r r a d i a t e d ascospores of Ophiostoma multiannulatum with a Coolidge tube at 50 K i l o v o l t s and 2-3 milliamperes f o r 100 minutes at a distance of 6-12 cm. and obtained mutant s t r a i n s which were unable to synthesize c e r t a i n chemicals necessary f o r growth.  As a result i t was decided that  a wide range of doses would be administered, s t a r t i n g at 50 r up to a maximum of 60,000 r .  Details of rate and duration of doses are shown i n  the following t a b l e . Radiation Experiment Voltage  -  90 K . V .  F.S.D.  -  15 cm.  Current  -  10 milliamperes  Rate  -  115 rontgens per minute  Filter  -  2 A l (H.V.L.2.3, Al) A l l treatments were continuous  Dosage (rontgens)  P.D.A. plates No. (duplicated)  Duration of a p p l i c a t i o n min. sec.  50  10  20  26  100  9  .19  52  200  8  18  1  43  400  7  17  3  25  600  6  16  5  9  800  5  15  6  52  1000  4  14  8  35  1200  3  13  10  17  1600  2  12  13  43  2000  1  11  17  10  - 45 Observations and Isolations The o r i g i n a l culture obtained from Dr. Martin i n Louisiana was l a b e l l e d i s o l a t e C f . B . This was a dark olive-green colony which produced p e r i t h e c i a , ascospores,  chlamydospores and conidia.  At f i r s t ,  ascospores and p e r i t h e c i a were produced c e n t r a l l y i n the oldest part of the culture, and l a t e r p e r i t h e c i a were found scattered randomly throughout the colony.  The p e r i t h e c i a had a diameter of about 100p.  was about 200JJ long and fimbriate at the t i p . diameter of approximately 3 ) i .  The neck  The ascospores had a  The hyaline, thin-walled conidia were  produced a e r i a l l y and endogenously i n f a i r abundance and measured about 12jax 6jjtin l e n g t h .  The chlamydospores were produced i n the medium,  at f i r s t endogenously and l a t e r by a process of budding.  They were com-  p a r a t i v e l y heavy-walled, olive-green i n c o l o r , r i c h with o i l droplets, and were usually pear-shaped but varied from short, club-shaped to almost spherical and sausage-shaped. The mycelium was f a i r l y uniform, branching abundantly and containing numerous o i l droplets. 6jJ.  The diameter of the mycelium was about  Seventy day o l d colonies growing at room temperature  had an average diameter of 6.6 cm.  (20-24°C.)  These colonies were dark oEve grey-  green and not uniform i n appearance.  There was no banding i n the  and p e r i t h e c i a and ascospore production were abundant.  cultures  Chlamydospores  were very few but the production of endoconidia i n the medium was f a i r l y widespread and surface conidia were produced i n great quantities i n a variety of shapes from simple c y l i n d r i c to clubbed and pear-shaped and even dumb-bell-shaped i n a few cases.  The rate of growth was f a i r l y  constant, being s l i g h t l y slower (2.2 cm.) during the i n i t i a l 5 days and  - 46 -  2.7 cm. for the l a s t 10 days (See table 7). Isolate IX  -  This "mutant" was i s o l a t e d from plate 7.  The o r i g i n a l i s o l a t e did not produce p e r i t h e c i a nor d i d i t produce any c y l i n d r i c c o n i d i a .  ;or ascospores,  Chlamydospores, however,  were produced and the mycelium was delicate and uniform.  Several  i s o l a t i o n s from fanfshaped sectors were made on potato dextrose agar plates (four plantings per p l a t e ) .  After about four days growth the cultures  were examined, and then from those which maintained t h e i r c u l t u r a l differences from the o r i g i n a l , transfers were made to four potato dextrose agar plates, making one planting per plate c e n t r a l l y . After 6 days growing at 27°C. the cultures were examined. average diameter of the cultures was 3 cm.  The  The cultures were completely  s t e r i l e , with slender i r r e g u l a r mycelium containing abundant o i l d r o p l e t s . The cultures were regular i n outline and l i g h t olive-green i n c o l o r . After growing f o r 70 days at room temperature the colony attained an average diameter of 5.6 cm., with the following c u l t u r a l c h a r a c t e r i s t i c s : The colony was dark olive-green with d e f i n i t e banding. white area i n the centre, 2 cm. i n diameter.  There was a r a i s e d  On the underside there  was a dark central core (2 cm. diameter), corresponding to the l i g h t raised area above, surrounded by concentric zones of a l t e r n a t i n g l i g h t and dark c o l o r .  This colony produced no surface c o n i d i a , but chlamydospores  were p l e n t i f u l i n the medium. i n i t i a l s were observed  Perithecia were absent and no p e r i t h e c i a l  i n the four platew examined.  S e r i a l transfers  were made and the f i f t h generation colony, 21 days o l d , was examined.  The  c h a r a c t e r i s t i c s remained true as described before except that the production of chlamydospores was g r e a t l y reduced.  Table 8 shows that at 27°C the  - 47 -  Table 7  Rate of Growth of Isolates on Potato Dextrose Agar at 1 0 ° C.  Diameter of growth i n cm. Plate No.  5th day  7th day  1 2  no growth ti  no growth it  0.6 0.3  IX  1 2  0.2 0.2  0.2 0.2  0.2 0.3  2X  1 2  0.3 0.4  0.3 0.4  0.3 0.4  4X  1 2  0.5 0.5  0.5 0.5  0.5 0.5  5X  1 2  0.5 0.5  0.5 0.5  0.5 0.5  6X  1 2  0.5 0.5  0.5 0.6  0.5 0.6  7X  1 2  0.5 0.5  0.5 0.5  0.5 0.6  8X  1 2  0.4 0.5  0.5 0.5  0.5 0.6  9X  1 2  0.6 0.6  0.65 0.7  0.7 0.7  10X  1 2  0.5 0.5  0.5 0.5  0.6 0.5  Isolate No. C. f . B.  25th day  - 48 Table 8 Growth Rate of Isolates on Potato Dextrose Agar at 2 7 ° C .  Culture No. C. f. B.  Plate No.  5th day  1 2 3 4  2.1 2.3 2.2 2.1 2.2  IX  1 2 3 4  2.2 2.2 2.3 2.2 2.2  2X  1 2 3 4  1 2 3 4 1 2 3 4  1 2 3 4  1 2 3 ft  2.2  4.4  •  •  2.3  4.07  2.2  5.15  6.5  1.9  6.0 1.8  6.5  2.4  7.5 7.5 7.6 2.60  7.53  4.9  5.5 6.5 6.5 6.5 2.1  6.5 4.6 4.5 4.5 4.4  2.2  1.8 1.6 1.7  4.5  7.4 7.0 7.0 6.6 2.3  3.47  4.7  -  -  1.7  7.0 6.2 5.7 6.0  3.6 3.5 3.3  -  1.7  2.7  6.2 7.3  5.5 5.0 5.1 5.0 2.6  7.6 6.5 6.5 6.5 6.5  3.5 4.5 5.0 3.3  2.3 2.1 2.4 2.1 2.2  7X  '  4.0 4.6 4.5 4.6  2.7  2.1 2.0 2.1 2.1 2.1  6X  4.9  2.5 2.5 2.6 2.6 2.55  5X  2.2  Av. d i f f . i n growth  7.3 7.5 6.6 8.0  4.9 5.0 4.6 4.9  2.3 2.2 2.3 2.2 2.25  4X  Diameter i n centimeters Av. d i f f . Av. d i f f . i n growth 10th day i n growth 15th day  2.0  5.97  4.2  - 49 Table 8 (Cont'd)  Diameter i n centimeters Culture No.  8X  Plate No.  Av. d i f f . 5th day i n growth  1 2 3 4  Av. d i f f . 10th day i n growth  1.75  2.6 2.8 2.6 2.5 2.62 10X  1 2 3 4  3.42  1.67  5.0 5.3 4.4 5.2 2.62  4.79  1.65  3.3  5.85  4.18  7.5 7.5 7.5 7.5 2.35  7.5  2.53  5.1 5.9 5.6 5.5  3.3 3.2 3.3 3.4  1.7 1.6 1.6 1.7 1.65  5.5 6.0 6.0 5.9  3.3 3.5 3.5 3.4  1.7 1.7 1.8 1.8  1.75  Av. d i f f . 15th day i n growth  1.65  5.52  3.87  fungus grew t o a diameter of about 6.5 cm. i n 15 days and that the rate of growth was f a i r l y constant, being only s l i g h t l y f a s t e r during the f i r s t 10 days than during the l a t t e r 5 days.  This s t r a i n i s s l i g h t l y slower  growing than the o r i g i n a l i s o l a t e B. From s t r a i n IX, three further i s o l a t i o n s (lXa, lXb, and l X c ) were made from sectors i n the colonies. lXa -  This i s o l a t i o n was made from a colony IX growing at 27°C on P.D.A.  The fan-shaped sector appeared very l i g h t , almost white, as compared with the r e s t of the colony.  The mycelium was sparse and almost  entirely  surface growing. Closer examination showed that the mycelium was slender, uniform and sparse with comparatively few o i l droplets and the individual c e l l s  - 50 -  were very long.  Neither p e r i t h e c i a l i n i t i a l s nor conidia were observed,  and only one chlamydospore was found. lXb -  This i s o l a t e was obtained from a. IX colony growing at 2 7 ° C . on  P.D.A.  This sector was very d i s t i n c t l y darker than the rest of the  colony. The mycelium was more abundant than i n l X a , varying from color-l e s s , i n the younger mycelium to olive-grey-green with age.  The mycelium  was not very uniform and contained more o i l droplets than did l X a .  The  c e l l s were shorter and the c e l l walls t h i c k e r than i n l X a . This i s o l a t e was completely s t e r i l e , producing neither sexual nor asexual spores.  The colony was slower growing than I X , a t t a i n i n g  an average diameter of 2.2 cm. i n 6 days at 2 7 ° C . lXc -  This i s o l a t e was made from a fam*shaped sector i n one of the IX  cultures.  It was s i m i l a r to the o r i g i n a l IX i n most respects except  that the colony was much l i g h t e r olive-green and i t s rate of growth o (an average of 3.5 cm. on P . D . A . i n 6 days at 27 C . ) was f a s t e r than that of the o r i g i n a l .  Peculiar anastomosing hyphae was observed i n mounts  made from the culture. 2X -  This i s o l a t e was obtained from a sector i n Plate 17.  The o r i g i n a l  culture was dark grey-green with a central abundance of p e r i t h e c i a and also a narrow (4 mm.) band containing p e r i t h e c i a at a distance of 2 cm. from the centre. no p e r i t h e c i a .  The sector was much l i g h t e r i n color and contained After 26 days growing at room temperature on P . D . A . the  i s o l a t e attained a diameter of 4.7 core, 2.2 cm. i n diameter.  The rest of the culture was a l i g h t e r color,  similar to the o r i g i n a l sector. seen.  cm. with a central dark olive-green  No perithecia nor chlamydospores were  Surface conidia and endogenous conidia i n the medium were abundant.  - 51 Seventy day o l d cultures growing under s i m i l a r conditions had an average diameter of 6.5  cm. (average of 4 p l a t e s ) .  The cultures  were l i g h t grey with no a e r i a l mycelium but contained abundant grey-white surface conidia.  No mature p e r i t h e c i a were observed, but a narrow (3 mm.)  band of p e r i t h e c i a l i n i t i a l s were seen at a radius of 1.5 centre. 2.5  cm. from the  Another d i s t i n c t continuous dark band was found at a radius of  cm. from the centre, the mycelium i n t h i s area being a darker ferey-  green with no p e r i t h e c i a l i n i t i a l s . A sixth generation c u l t u r e , 6 days o l d , grown on P . D . A . had a diameter of 3.5  cm.  This culture contained numerous p e r i t h e c i a , no  chlamydospores, and abundant surface conidia and conidia produced endogenously i n the medium.  This culture suggests some reversion to the  o r i g i n a l C . f i m b r i a t a , i s o l a t e B. 4X -  This i s o l a t e was obtained from a sector on Plate 17.  The sector  contained an increased number of p e r i t h e c i a and p e r i t h e c i a l i n i t i a l s . A seventy day old culture grown on P . D . A . at room temperature produced no p e r i t h e c i a , but there were numerous p e r i t h e c i a l i n i t i a l s .  There was  a p r o l i f i c production of surface conidia but comparatively few conidia i n the medium and few chlamydospores.  The colony was dark olive-green,  showing vague banding with hyaline and pigmented mycelium. had an average diameter of 6.3  cm.  From t h i s i s o l a t e a sub-isolate  made from a sector i n one of the c u l t u r e s . 4Xa -  The colony was  This was l a b e l l e d 4Xa.  Twenty-one day old cultures of 4Xa growing on P . D . A . at room  temperature had an average diameter of 3.3  cm.  The colony was a l i g h t  olive-green i n color i n the medium but the surface was much l i g h t e r grey with a mealy consistency.  No p e r i t h e c i a were observed.  Conidia were  more numerous than i n 4X and chlamydospores were p l e n t i f u l .  - 52 -  A comparison between 6 day old. cultures of 4X and 4Xa growing on P . D . A . at 2 ? ° C . showed that there were no p e r i t h e c i a nor p e r i t h e c i a l i n i t i a l s i n the 4X cultures, whereas the 4Xa cultures contained a copious amount of p e r i t h e c i a l i n i t i a l s . ; ' 4X cultures appeared to produce conidia only a e r i a l l y , whereas the 4Xa culture produced conidia both by budding and endogenously from conidiophores. 9X -  No chlamydospores were observed i n any of these c u l t u r e s .  This i s o l a t e was made from a culture of C. fimbriata B, treated  with 50 r . continuously over a period of 26 seconds.  The sector which  was observed 3 weeks after i r r a d i a t i o n , was grey-green i n color on the edge of the colony.  Immediate examination showed that  chlamydospores  were p l e n t i f u l and the endogenous production of chlamydospores and conidia was widely d i s t r i b u t e d .  Many mature perithecia were observed.  Seventy day old cultures growing on potato dextrose agar at room temperature had an average diameter of 7.7 cm.  This i s o l a t e was  d e f i n i t e l y f a s t e r growing than the other i s o l a t e s , and the cultures showed a very d e f i n i t e banding. These cultures produced no p e r i t h e c i a . f a i r l y numerous and conidia were few. pigmentation being observed.  Chlamydospores were  The mycelium was hyaline, no  The mycelium was not very uniform i n width  and d i d not appear to be very vigorous but contained abundant o i l droplets. F i f t h generation cultures growing on P.D.A. at room temperature attained an average diameter of 4.2 cm.  These cultures maintained the  characters described previously. 8X -  This i s o l a t e was made from a culture of C_. fimbriata B, which was  not treated with X - r a y s .  Sectoring was observed on the culture and  - 53 i s o l a t i o n s were made.  Simultaneous observation indicated the  presence  of a dumb-bell shaped b a c t e r i a l contaminant widespread throughout the medium.  This contaminant was eliminated by adding a c e t i c acid to the  medium to reduce the pH. The sector was brown-grey i n c o l o r , i n contrast to the greygreen of the parent c u l t u r e .  The conidia were produced copiously, and the  conidia were observed to be more slender and longer, on an average, than those of the parent culture ( 1 2 / x - 14/A.).  NO chlamydospores were observed  and p e r i t h e c i a were absent. F i f t h generation cultures growing on potato dextrose agar at 27°C. f o r 6 days had an average diameter of 3.3 cm. and the were s i m i l a r i n many respects t o the o r i g i n a l .  cultures  The colony did not maintain  i t s brown-grey color but was not very l i g h t olive-green, much l i g h t e r than the o r i g i n a l .  The mycelium too, was l e s s pigmented, almost  colorless  i n appearance, weak and i r r e g u l a r i n growth.  Conclusions From the tables i t can be seen that i s o l a t e s B and 9X had about the same rate and amount of growth.  At f i r s t , the average increase i n  r a d i a l growth was higher i n 9X ( 2 . 6 2 ) as compared to that of i s o l a t e B (2.2).  In the l a t e r stages, however, i s o l a t e B had a greater average  increase i n growth. constant. f i v e days.  The rate of growth of i s o l a t e IX seemed to be f a i r l y  Isolate 9X grew the fastest of a l l the cultures i n the  first  The rate of growth of i s o l a t e A increased with time u n t i l the  f i f t e e n t h day.  In general, during the l a s t five days most of the i s o l a t e s  increased t h e i r rate of growth.  - 54 FURTHER IRRADIATION STUDIES  Because of the wide range of rontgen units administered to induce mutations i n plants ranging from 50 r . for p o l l e n grains (Deschner and Sparrow, 60) to over 50,000 r . f o r B a c i l l u s c o l i spores (Duggar, 61), i t was decided to increas the dosage on the fungus under study i n an attempt to observe the effect of t h i s more d r a s t i c treatment and to determine the amount of X - r a d i a t i o n that i s l e t h a l to the organism.  Materials and Methods The medium employed here was modified Czapek's Agar, the same as that used e a r l i e r i n t h i s study.  Conidia and ascospores i n a water  suspension were spread out on s t r i p s of d i a l y z i n g membrane and placed c e n t r a l l y on the P e t r i plate as dewcribed previously.  The spores were  i r r a d i a t e d one-half hour a f t e r being taken from the cultures and placed on the membranes.  The s u p e r f i c i a l X-ray unit was used at 10 milliamperes  and 90 K i l o v o l t s .  A 0.5 mm. aluminum f i l t e r was employed to eliminate  extraneous rays.  The distance between the source of the rays and the  target was 15.9 cm.  In the c a l c u l a t i o n of the doses compensation was  made f o r the P e t r i dish l i d which was l e f t i n p o s i t i o n during treatment. The doses were delivered continuously at a rate of 375 rontgen units per minute.  Results and Observations It was estimated that about 60$ of the spores germinated after Treatment 1.  From transfers made of the treated spores to other p l a t e s ,  three 'mutants' appeared.  These were observed microscopically, i s o l a t e d  and l a b e l l e d 55Xa, 55Xb, and 55Xc.  - 55 -  Dosage i n Rontgen Units  Treatment  Duration of Treatment i n Minutes  1  20,625  55  2  48,000  128  3*  50,000  90  4  (control)  * A three day o l d culture treated without 0.5 mm. aluminum f i l t e r . Isolate 55Xa was completely s t e r i l e , producing white a e r i a l mycelium and microscopically very s i m i l a r to i s o l a t e IX obtained a f t e r treatment with 400 r . Isolate 55Xb appeared as a darker olive-green sector, as compared to the o r i g i n a l C f . B , and contained numerous c o n i d i a , both on the of the medium and i n the medium. were observed.  surface  Neither chlamydospores nor p e r i t h e c i a  The mycelium was vigorous and uniform.  Isolate 55Xc produced p e r i t h e c i a , ascorspores and both conidia and chlamydospores i n abundance.  The sector was markedly darker than the  parent colony and darker than sector 55Xb.  Neither of the i s o l a t e s 55Xb  and 55 Xc remained true to the change but reverted to the o r i g i n a l . Spores i n treatment 2 showed very low percentage germination even after 23 hours. below 1%.  It was estimated that the percentage germination was  Percentage estimates were made from counts of the number of  germinated and ungerminated spores i n twelve random and separate under the low power (xlOO). germinated spores.  fields  No growth resulted from transfers made of  - 56 The 3 day old culture used i n Treatment 3 was 2.5 cm. i n diameter and contained large quantities of c o n i d i a , butneither chlamydospores not perithecia were present.  A high dosage was administered i n an attempt  to estimate the l e t h a l effect of r a d i a t i o n .  The appearance of the  culture after i r r a d i a t i o n was markedly different from that of the untreated C f . B cultures. The colony attained a diameter of 7 cm. after 30 days growth at 27°C  The central (2.5 cm.) area was markedly darker than the rest of the  culture, having a dark brown-black c o l o r . conidia and perithecia with ascospores, chlamydospores were present.  This area contained numerous  e s p e c i a l l y at the centrej a few  The mycelium here appeared d e f i n i t e l y darker  and more vigorous than that outside t h i s l o c a t i o n .  On the immediate  periphery of t h i s area there was a d i s t i n c t complete band (5 mm. wide) of perithecia bearing gelatinous masses of ascospores on t h e i r fimbriate tips.  Beyond t h i s band, the colony consisted of dark.mycelium (but  l i g h t e r than that i n the central core) and more abundant but smaller perithecia i n scattered c l u s t e r s .  Chlamydospores were obviously more  prevalent here than i n the central dark a r e a .  No 'mutants' have been  observed from sub-cultures.  Conclusions 1.  Vegetative growth appears much more, resistant to i r r a d i a t i o n than spores.  2.  Apparently i d e n t i c a l mutants seem to occur independently under different  3.  treatments.  Comparatively, t h i s fungus i s very resistant to the l e t h a l of X - r a d i a t i o n .  effect  - 57 4.  An approximate estimate of c u l t u r a l changes show about 2$ mutations, about 1.5$ reversion and about 0.5$ remained true to change f o r about 4 months.  5.  Reversion occurred most often during the f i r s t two generations of sub-cultures.  However, one culture which appeared stable f o r  three months reverted to the o r i g i n a l . 6.  Continuous growth on the same medium (P.D.A.) appears to induce degeneration i n the  7.  isolates.  After 4 months 'mutant' i s o l a t e s produce subsidiary "sports" spontaneously i n culture.  S i x such i s o l a t e s were i n d i s t i n g -  uishable morphologically from i s o l a t e s already made. 8.  As a r e s u l t of t h i s i n s t a b i l i t y i n the cultures i t i s proposed that a l l i s o l a t e s be termed  'mutants'.  It has been found that i n mutation work the agar colony technique has the l i m i t a t i o n of permitting detection only of those colonies whose mutation i s r e f l e c t e d by conspicuous change i n c u l t u r a l or morpho l o g i c a l appearances.  Probably a f a r greater number of physiological mutants  are produced that vary i n one or more biochemical properties but are morphologically indistinguishable from each other and from the parent c u l t u r e , both micro- and macroscopically.  Further studies i n the  selection  of culture media i s necessary for the detection of biochemical mutants. The chemical basis f o r reversion of cultures i s s t i l l not c l e a r . Steinberg and Thorn (62) suggested that some mutations, at l e a s t , might be due to a l t e r a t i o n s i n c e l l p r o t e i n (chromatin, enzymes e t c . )  of the  fungus, and further suggested that reversion consisted of r e p a i r of chemical i n j u r y to c e l l p r o t e i n .  This suggestion lends i t s e l f  to the  speculation that the gene may be present and i n t a c t but modified to require altered conditions of operation.  This view i s further supported  by the fact that Stokes (63) observed that an X-ray induced pyridoxineless s t r a i n of Neurospora s i t o p h i l a grew normally on an acid medium with ammonium-nitrogen.  - 59 THE EFFECT OF ULTRA-VIOLET RAYS ON SPORES OF OPHIOSTOMA FIMBRIATUM  P e t r i plates containing s t e r i l e d i a l y z i n g membrane were prepared as described before.  Conidia and ascospores, i n a d i l u t e suspension were  placed on the membrane sections and also d i r e c t l y on the agar surface. These were treated i n a s t e r i l e chamber with a G . E . Sterilamp at a distance of 3 inches from the source of the l i g h t , the l i d s of the plate being removed.  The plates were treated f o r 5» 10, 15, 20, 30, 40, 45 and 60  minutes.  Observations The majority of spores, e s p e c i a l l y the c o n i d i a , appeared lysed with treatments over 10 minutes duration.  The collapsed remains of these  spores were strewn a l l over the surface of the medium and on the membrane. Germination was very poor when the treatment exceeded ten minutes but the effect on the germination of the ascospore was l e s s than that on conidium germination at 15 minutes treatment.  Ascospores d i d not appear  lysed even though germination was unimpaired.  There was no germination  i n plates treated over 20 minutes.  One ascospore seemed to have germinated  a f t e r 40 minutes treatment but on t r a n s f e r to another plate the spore was apparently k i l l e d , as no further growth took place. Transfers were made from plates treated for 5, 10 and 15 minutes for further growth.  Only one i s o l a t e  from a 5 minute-treated c u l t u r e . a previous i s o l a t e , 9X.  (16X) was obtained and t h i s was made  So f a r , 16X appears to be i d e n t i c a l to  - 60 -  The Cellophane Membrane Method The cellophane membrane method of spore germination f o r mutation work has several  advantages.  First, i t facilitates  the transfer of germinated spores.  Thus,  by t r e a t i n g . a single plate containing several pieces of membrane the spores can be 'thinned* out or dispersed by transferring on to other plates.  This increases the p o s s i b i l i t y of growth and the appearance of  mutants by reducing the competition i n growth with the parent 'wild type*. This method also f a c i l i t a t e s  r e l a t i v e l y easy c y t o l o g i c a l  study  of i r r a d i a t e d , germinated or ungerminated spores, or the cytology of the i r r a d i a t e d growing germ tube.  On suitable media the germination can be  followed d i r e c t l y , the organism obtaining nutrients and growing without becoming included i n the medium.  The spores thus remain clean and  uncontaminated for staining purposes.  With some care the membrane bearing  the spores could be put through the k i l l i n g and f i x i n g solutions,  alcohols,  s t a i n s , and even a c i d hydrolysis without much l o s s . By t h i s method germinating spores could be subjected to  several  different nutrient treatments merely by t r a n s f e r r i n g the germinating spores on the membrane. Instead of several different i r r a d i a t i o n treatments of smaller amounts of organisms t h i s method provides f o r the treatment of a large quantity which can be 'thinned' out l a t e r .  This favors greater uniformity  i n treatment. This process could also be used for single spore i s o l a t i o n , espe c i a l l y for spores that are comparatively small and where germination i s necessary before i s o l a t i o n .  Several (1-2 dozen) small squares (1-2 mm^)  - 61 -  of s t e r i l e membrane paper are placed on an appropriate nutrient media, a suitable d i l u t i o n of the spores i s poured on the plate over the membrane sections.  At the desired stage of germination a single spore  isolation  can be made by removing a membrane section containing one spore and r e planting on another p l a t e .  - 62 -  PATHOGENICITY STUDY  Ophiostoroa fimbriatum i s known to be the causal organism of the black rot disease of stored sweet potato (Ipomoea batatus).  It was  decided that the various i s o l a t e s be subjected to a pathogenicity t e s t to observe t h e i r r e l a t i v e degrees of virulence on the susceptible  host  tissues.  Materials and Methods A nondescript variety of sweet potato was obtained from a l o c a l grocery store.  The sweet potatoes were thoroughly washed and cut into  sections, roughly 5 x 5 x 3 'skin  1  inches.  Care was taken t o r e t a i n the epidermal  on one large surface of each section.  Two of these sections were  placed into each of twenty Erlenmeyer flasks against the wall i n a semiupright p o s i t i o n with the epidermis upwards. 10 cc. of water.  To each flask was added  The f l a s k s were stoppered with cotton and s t e r i l i z e d  i n the autoclave. After s t e r i l i z a t i o n the t i s s u e s i n the flasks were 1 inoculated with 10 different i s o l a t e s of the fungus, each block i n two f l a s k s being iisnoculated with the same i s o l a t e .  The xhnoculations were made by deposit-  ing the .'..inoculum i n a needle break i n the epidermis of the potato block to simulate natural jimoculations.  The f l a s k s were incubated at room  temperature.  Observations Both d i r e c t and microscopic observations were made to determine not only the pathogenicity but also the c u l t u r a l c h a r a c t e r i s t i c s of the  - 63 i s o l a t e s growing on t h i s natural substrate. The i n f e c t i o n on the skin caused by i s o l a t e I X , consisted of a few r e l a t i v e l y large raised postules. postules was white. coloration.  The a e r i a l mycelium on these  The i n f e c t i o n i n the i n t e r n a l tissue produced a grey  The centre of the r a i s e d postules consisted of a mass of  dark olive-green mycelium.  The cultures were completely s t e r i l e .  Neither  asexual conidia, chlamydospores nor sexual spores were produced i n the lesions.  Comparatively, destruction of the potato tissue was not very  great. In the 2X cultures there was no surface mycelium but instead there was a luxuriant production of hyaline conidia. present but were r e l a t i v e l y very few.  Chlamydospores were  The colonies were grey and consisted  of several small postules scattered over the epidermis of the  sections.  Mycelium was abundant and vigorous i n the tissues of the tuber.  No  perithecia were observed i n these cultures. The i s o l a t e 4X appeared to be a very v i r u l e n t i s o l a t e .  Con-  s i s t e n t l y , a l l four blocks i n the two f l a s k s l o s t t h e i r o r i g i n a l conformation and became a soggy grey mass. and abundant.  The mycelium was dark olive-green, vigorous  Copious amounts of conidia and chlamydospores were present  and perithecia and ascospores were produced i n i s o l a t e d areas over the tissues. The i s o l a t e 5X appeared to be one of the l e a s t v i r u l e n t Conidia were extremely p l e n t i f u l , especially on the surface. were present but very scant.  isolates.  Chlamydospores  P e r i t h e c i a and ascospores were a l s o present.  The shape and color of the sections was not destroyed except i n the necrosed areas.  Several small raised postules were produced on the  - 64 -  epidermis of the sweet potato sections innoculated with i s o l a t e 6X. appeared to be a r e l a t i v e l y v i r u l e n t s t r a i n . olive-green on the surface.  This  The mycelium was dark  The inside of the pustules consisted of an  almost black mass of vigorous pigmented mycelium producing large numbers of dark olive-green chlamydospores.  Conidia were p l e n t i f u l and the  cultures were teeming with p e r i t h e c i a and ascospores. Isolate 7X was s i m i l a r i n c u l t u r a l c h a r a c t e r i s t i c s to oX but was much l e s s v i r u l e n t . Isolate 8X was obviously the most v i r u l e n t i s o l a t e .  The fungus  involved the e n t i r e epidermal and c e n t r a l t i s s u e s of the sections, bringing about complete destruction.  Conidia and chlamydospores were p l e n t i f u l  but p e r i t h e c i a l production was scant. Large raised white l e s i o n s were produced by i s o l a t e  9X.  These  consisted of very good mycelial growth but l i t t l e penetration into the c o r t i c a l tissues was observed.  Conidia were i n great numbers.  spores, though p l e n t i f u l , were not f u l l y developed.  Chlamydo-  The chlamydospores  were pear-shaped but thinner-walled and smaller and contained much l e s s pigment than the t y p i c a l dark grey-green heavy-walled spore of t h i s type. The mycelium was abundant, slender, hyaline, containing no pigment.  The  dark color of the sections around the l e s i o n s appeared to be due to oxidation of the host t i s s u e s .  No perfect stage was observed i n t h i s  culture. Both sexual and asexual spores were present i n abundance i n cultures os i s o l a t e 10X.  The mycelium were heavily pigmented and appeared  as a dark mass. Isolate 15X was not very v i r u l e n t and was comparable to  isolate  - 65 IX and 9X i n virulence.  S t r i k i n g l y , no conidia were produced but a few  chlamydospores were found i n the l e s i o n s . green and vigorous.  The mycelium was dark grey-  On one of the potato blocks a » s p o r t ' appeared as  a raised white cottony mass.  Isolations were made from t h i s and observa-  tions revealed that t h i s portion of the culture was completely  sterile  and produced l i g h t , hyaline, slender mycelium i n contrast to the  vigorous  pigmented mycelium of the o r i g i n a l c u l t u r e . In order of ascending pathogenicity the strains appeared as follows:  7X, 5X (9X, 15X, IX) 1CX, 2X, 6X, 4X and 8X.  The degree of  pathogenicity was determined by v i s u a l comparison based not on the rate but rather the amount of destruction of sweet potato t i s s u e .  The l e s i o n s  varied from l o c a l i z e d and r e s t r i c t e d infections to those i n which the entire tissue sections were involved. Apart from the sectional conclusions already made the following general comments can be added.  It appears, from an o v e r a l l study,  that  the hyaline c y l i n d r i c conidia are the type of spores most e a s i l y produced i n culture.  Chlamydospores and perithecia are more r e l u c t a n t l y formed  and t h e i r production seems to be correlated to the degree of pigmentation of the  cultures. The conidia are produced i n two ways.  The endogenous production  of conidia has been observed to occur only within the media.  The  conidiophore i s only s l i g h t l y different from an ordinary mycelial strand. The terminal producing c e l l i s l a r g e r and tapered toward the t i p with a basal c e l l that appears bulbous. single conidiophore.  Several conidia are produced from a  The second method of conidium formation i s by the  fragmentation of the a e r i a l mycelium and was observed to occur on the  - 66 surface of the substrate.  In most cultures what appeared to be surface  mycelium broke i n t o innumerable u n i c e l l u l a r fragments (conidia) by the touch of a needle's point.  The two methods of conidium formation appear  to be related to the difference i n the degree of aeration i n the two s i t e s and also to the osmotic i n t e r r e l a t i o n s h i p s between the medium and the fungus hypha. The other asexual spore i s referred to i n t h i s study as a chlamydospore since i t has a r e l a t i v e l y heavy w a l l , contains stored food i n the form of o i l or f a t and was seen to be produced, not only l a t e r a l l y and t e r m i n a l l y , but also from the i n t e r c a l a r y regions of the mycelium (see p . 23). genously,  Although the majority of these spores are produced endo-  some have been observed to be produced by a process of budding  (Plate-: 1 ) .  These spores are always produced within the media.  In a l l  the l i t e r a t u r e reviewed, these spores are alluded to as c o n i d i a , and there i s no mention of chlamydospore formation by budding nor the production of conidia by fragmentation. The degree of pigmentation of the mycelial c e l l s seems to be correlated to the vigour of the c u l t u r e s .  It appears, a l s o , to  the degree of pathogenicity of the i s o l a t e s . c e l l s of l e s s pigmented hyphae.  affect  O i l globules accumulate i n  The pigment i n t h i fungus i s retained  within the c e l l s and does not diffuse out into the medium.  Cultures of  i s o l a t e B grown i n the dark and i n l i g h t showed no apparent difference i n the degree of pigmentation. Two i s o l a t e s which do not produce p e r i t h e c i a and sexual were planted on the same p l a t e .  spores  No sexuality resulted from the meeting of  the hyphae, suggesting e i t h e r the i n c o m p a t i b i l i t y of h e t e r o t h a l l i c  strains  - 67 or the i n a b i l i t y of the single or mixed i s o l a t e cultures to produce sexual bodies under those conditions of growth. It i s also t o be noted that •sports' s t i l l a r i s e from the sixth and seventh generation sub-cultures of the o r i g i n a l i r r a d i a t e d spores. There a r e , however, many reversions. Isolate B haw been found to be i n h i b i t e d i n growth by a species of P e n i c i l l i u m .  A P PEN D I X  PLATE I - (a) Chlamydospore i n the process of budding and (b) terminal hypha producing a pear-shaped chlamydospore.  - 68 -  PLATE I  PLATE I I - (a) Diagrammatic representation of the endogenous production of the hyaline conidia and (b) the helmet-shaped ascospores.  - 69 -  PLATE I I  a  b  o  PLATE I I I . Stages i n the d i v i s i o n of the nucleus of the germinating conidia. ( I ) The resting nucleus. (II) The transformation of the nuclear sap into chromatin strands, (III) Inception of the germ tube i n i t i a l s along the t h i n areas of the c e l l w a l l . (IV) Only one germ tube i n i t i a l continues growth, random separation of chromatin material taking place, note denser cytoplasm at germ tube t i p . ( V ) Migration of ghromatin material. (VI) Second nuclear d i v i s i o n . (VII) as i n (V). (VIII) Third nuclear d i v i s i o n and the inception of the f i r s t septum. (IX) A l a t e r stage of (VIII), showing completed cross-walls and the f i r s t d i v i s i o n of the t i p nucleus.  PLATE IV, Photomicrographs showing abnormal and germination of the sexual spores of 6. fimbriatum treated with c o l c h i c i n e . (I) The formation of t h i n walled, pear-shaped bodies l a t e r a l l y on the vegetative hypha. ( I I ) A s i m i l a r type of body formed from a conidium. ( I l l ) Another abnormal structure formed from a conidium. (IV) Abnormal conidium. (V) Unusual spore from a conidium. (VI) as i n (II) but with further budding. (VII-IX.) Terminal, l a t e r a l and i n t e r c a l a r y formation of chlamydospores. For further d i s c r i p t i o n see text.  PLATE V . The effect of various sources of nitrogen on the rate of growth of cultures of 0. fimbriatum. A l l cultures grew on an equivalent of 0.76$ nitrogen, ( l ) Asparagine. (2) C o n t r o l . (3) Potassium n i t r a t e . (4) Ammonium sulphate. A l l cultures 16 days o l d .  - 72 -  PLATE V  PLATE VI. Small aquares of d i a l y z i n g membrane placed on media and ready f o r planting. (Ixa) A culture of t h i s i s o l a t e showing a further fan-shaped 'mutant' sector. ( l 6 ) and (4) Comparison of the growth and color c h a r a c t e r i s t i c s between i s o l a t e s 16X and 4X. (Note i s o l a t e 16X on dark background).  - 73 -  PLATE VI  PLATE VII. Cultures of 0. fimbrlatum growing on d i f f e r e n t media at various pH. ( l ) Growth on sweet potato dextrose agar at pH $.0. (2) on potato dextrose agar at pH 3.9. (3) on beet dextrose agar at pH 5.0.(most extensive growth) and (4) on parsnip dextrose agar at pH 3.9.  - 74 -  PLATE VII  X-RAY  TREATMENT  BETWEEN  DOSAGE TIME  2  IN  OF  4  SHOWING RONTGEN  RELATIONSHIP  UNITS  AND  ADMINISTRATION  6 TIME  THE  8 IN  MINUTES  10  12  14  16  - 76 -  THE  SUPPOSED  OF  HO  OXIDISED  ROLE  OF  MELANIN  COPPER PIGMENT  IN  THE O.  FORMATION  FIMBRIATUM  TYROSINE  rCHiCH-COOH *i NH  C H ,  IN  TYROSINASE -TYROSINE  C H - C O O H - C u *  NH,  -CUPROUS  E N Z - C i A O ,  1  COMPLEX  TYROSINASE (REDUCED)  Redox ENZ-Ci/  CHCOOH NH.  Q  I  J  CHCOOH-Cu*  EN2-  NH,  Redox  T Y R O S I N A S E (OXIDISED)  3.4-DIHYDROXY P H E N Y L A L A N I N E (DORA)  TYROSINASE-DOPAQUINONE CUPROUS  COMPLEX  I CHCOOH  o  DOPAQUINONE  NH,  Polymerization  I  OXIDISED  MELANIN  - n BIBLIOGRAPHY 1.  Halsted, B.D.  Some fungus diseases of the sweet potato.  Expt. S t a . B u l l .  76: 32.  Sylloge fungorum.  1890.  2.  Saccardo, P . A .  3.  Stevens, F . L . 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