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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 Bri 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 this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1957 - i -ABSTRACT The fungus Ophiostoma fimbriatum (E & H) Nann, though exception-a l l y rare i n northern climates, i s f a i r l y widespread i n tropica l and sub-tropical areas of the world causing diseases on many species of plants. The disease producing capabil i t ies 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 interesting from the point of view that i t produces, very readily on sweet potate dextrose agar, two types of ; asexual or vegetative spores and the perfect stage with the perithecia containing ascospores. Several strains of the fungi have been isolated from natural habitats. This work deals, i n general, with a study of the gross morphology of th is ascomycete and some observations on the nuclear apparatus of the resting and germinating conidia. More speci f ica l ly , this study treats with certain factors i n nutri t ion which affect the physiology i n such a way that the growth and sporulation characteristics of the organism are altered. Since several strains of 0 . fimbriatum have been isolated naturally i t i s thought that these must have been derived from mutant changes occurring i n an orig inal "wild" form which was propagated to more susceptible varieta l hosts. As a resul t , studies are undertaken i n an attempt to induce changes i n an or ig inal 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 ascertain the relat ive degree of virulence between the new-formed strains. - i i » This work shows that the cultural characteristics and repro-ductive behaviour of this fungus could be modified by specific variations i n the culture medium. It i s shown among other things, that copper, i n the role of a micro-nutrient, plays a definite part i n the manifestation of sexuality and i n the development of pigmentation in the organism. "Mutations" are produced by using X - i r r i d i a t i o n and u l tra-v io le t rays as inductive agents. Many of the new-formed "mutants" are unstable and back mutation to the original "wild" type i s common. In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree th 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 reference and study. I f u r t h e r agree that permission for. extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s understood tha t 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 f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permission. Department of & ^tgy^Aj n^yvd fcjjL The U n i v e r s i t y of B r i t i s h Columbia, Vancouver S, Canada. Date QdjLptt^&j^ l9f - i i i -TABLE OF CONTENTS Page Abstract i Table of Contents i i i Acknowledgments i v Review of Literature 1 ( i ) Class i f icat ion 1 ( i i ) Sexuality and Strains 2 A Morphological Study 8 A Cytological Study 13 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 tra-v io le t rays 41 Further Irradiation Studies 54 The effect of u l tra-v io le t rays on spores 59 Pathogenicity Study 62 Appendix".'..^ 6g Bibliography 76 - i v -ACKNOWLEDGMENTS In the compilation of th i s work, I have received helpful 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 atthis University for his help and patient guidance and also to the late Dr. D.C. Buckland for the use of the equipment and materials i n his Forest Pathology Laboratory for the accomplishment of a part of this work. Thanks to Dr. J . S . Lot t , Radiologist, and Mr. Maurice Bullen, Research Physicist , 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 Agricultural Experimental Station at Baton Rouge for the isolate CeratostomeHa fimbriata B, which was used exten-sively i n this study. Thanks too, to Miss E l l e n Sprinnget for her technical help and for the typing of this thesis . REVIEW OF LITERATURE ( i ) Class i f icat ion 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-disintegration of the ascus walls and the l iberat ion of the ascospores into the cavity of the mature perithecia, 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. The pycnidia possessed a rostrum 20-30^ long and apical ly fimbriate. He found that the conidia were globose-el l ipt ic 5-9^ long. The fungus grew well on sweet potato producing dark, almost black, spots on the skin. 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 ight colored t ips arose from the medium. From these, Stevens thought, hyaline conidia were produced endogenously. Lehman (4) i n 1918 distinguished two types of conidia i n Sphaeronema fimbriatum. These he termed "hyaline conidia" and "olive conidia". 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 in Ceratostomella fimbriata in the Ceratostomatacae of the Sphaeriales. In 1932 Nannfeldt (6) removed the fungus from the Ceratostomatacae and placed i t in 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) in 1947 created another "class" of the "Ascomycetales" which they called "Class Haerangiomycetes". In this class they placed Ophiostoma and Melanospora in which the asci do not possess a definite cell wall but merely a plasma membrane or where the ascus wall is almost immediately dissolved after its formation. In these fungi, therefore, the ascus does not exercise its normal function of ascospore dispersion but these spores are carried out through the ostiole in a mass of "mucus" and rest in a drop in 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 in which definite functional asci occur. (ii) Sexuality and Strains Elliott (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 in 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 is not certain but in favour of this, Elliott found that perithecia - 3 -were always formed in groups, usually crowded together in 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 cell, an ascogonium, and a trichogyne. The trichogyne was continuous with the ascogonium; there was no dividing cell wall and this structure had no nucleus. The ascogonial nucleus, at this time, was very prominent. The trichogyne and antheridium fused at whatever point they came in contact with one another. The uninucleate upper cell 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 in the perithecial cavity. Eight ascospores arose in each ascus, and ascus walls, ascogenous hyphae and the remainder of the 'nurse cells' underwent autodigestion. Gertrud Mittman in 1932 (9) contrary to Elliott's report, failed to find any antherid. According to her, the ascogonium began as a single, uninucleate, somewhat curved terminal cell of a short lateral branch. This cell 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 , thought that i t was strange that the oogonium should display such intimate contact with the antheridium for apparently a fusion of the two structures rarely 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 Faull for Laboulbenia chaetophora Thax. (11). Andrus and Harter further stated that the oogonium by i t s division and by further division of i t s progeny, formed a multitude of cells a l l destined to become asci. They observed the complete absence of any hyphal connection between the body of the perithecium and the ascogenous ce l l s which occupy i t s interior. They thought this situation occurred because these cells 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 original protoplast i n the sub-terminal c e l l of the ascogone. These cells were imbedded in a nutrient medium which obviated the need for connecting hyphae. Andrus and Harter (10) confirmed Mittman as to the absence of croziers and as to the unwalled condition of the asci i n C. fimbriata. They further reported that the f i r s t nuclear division i s characterised by the development of a distinct ascus inside which, they suggested, seemed to be the membrane of the fusion nucleus. They considered that a l l three nuclear divisions occurred within this vesicle. Andrus and Harter further maintained that this vesicle expanded during nuclear division 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 asc i i n the ir younger stages are without a definite wall". Later work by Andrus and Harter (12) i n 1937, confirmed their ear l ier views and they further concluded that i n the i n i t i a t i o n of the a sc i , not only direct and indirect types of cleavage occurred, but also that the typica l crozier 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 iv is ion two comma-shaped and two bilobed bodies were found. These authors reported no reduction div is ion i n the ascus. Gwynne-Vaughan and Broadhead (13) observed a typical ascomy-cetous development of the f ru i t ing body, contrary to the findings of Andrus and Harter (10). They fa i l ed to confirm the absence of ascogenous hyphae and ascus walls, as reported by Andrus and Harter and could find no evidence of the ascus vesicle figured by Andrus and Harter and Mittmann i n the ascus. They found that the asc i developed by typica l crozier formation. Mittmann, i n 1932, demonstrated the homothallic nature of Geratostomella fimbriata. Olson, however, i n 1950 (14) succeeded i n i so lat ing heterothallic strains of this organism. Gaumann and Dodge (15) stated that there seemed to be a sl ight suggestion of heterothallism since antheridia and oogonia arose from separate hyphal strands. The antheridium coi l s about the ascogonium, which i s differentiated into 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 isolated and studied ten strains of Ceratostomella  fimbriata from lesions on sweet potato. He found that they differed i n pathogenicity to their suscept, i n morphology and compatibility. The ascospore progeny of four s e l f - f e r t i l e (perithecial) strains included not only the parental s e l f - f er t i l e strains but also their respective se l f - s ter i l e (non-parental) strains. Single conidium isolates which he made from monospore cultures were of the parental type. Olson also found that long-necked perithecia developed on the l ine between se l f - f er t i l e and se l f - s ter i le strains as well as between some of the l a t t e r . It was found that isolates from another strain produced no perithecia either alone or in combination with other se l f -steri le strains. The ascospore progeny of perithecia formed by combinations of strains comprised either the parental types alone, or^both parental and recombination types, suggesting that peri thecial production i s the result of at least two independently inherited genes. Ascospores from a homothallic s train gave r ise to heterothallic strains . The addition of 5JJ g thiamine to Czapek's medium was essential for perithecial production by isolates which were s e l f - f er t i l e on potato dextrose agar. However, the addition of this growth substance to the la t ter substratum did not induce the formation of perithecia i n naturally se l f - s ter i le strains. Olson also found that s e l f - f er t i l e cultures of Ceratostomella  fimbriata isolated from Hevea rubber trees i n Mexico and sweet potato from the United States, were similar morphologically but were not cross pathogenic. Both strains gave rise to se l f - f er t i l e and se l f - s ter i l e strains among single ascospore cultures isolated from them. He spermatized week-old cultures of se l f - s ter i l e sweet potato strains with spores from - 7 -s e l f - f e r t i l e rubber cultures and found that perithecia developed i n three days. Ascospores from these perithecia 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 isolations from the two cultures produced s e l f - f e r t i l e and se l f - s t e r i l e strains with the same pathogenicity as the parent. Hybridization occurred i n the experiment between the strains of Ceratostomella fimbriata isolated 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 factors for each sex. The presence of both re-sulted i n hemaphroditism. Pontis (17) made cross ^inoculation experiments using the patho-gens 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 isolated and incubated at room temperature. At the same time coffee trees were iusoculated with C. fimbriata from sweet potatoes. None of these cross .inoculations produced infection, indicating that the two diseases are caused by different strains of the same fungus. A MORPHOLOGICAL STUDY The germination, growth and development of a single conidium was followed. The conidium in i t ia ted germination on sweet potato medium at room temperature (24°C - 26°C) four and a half hours after i so lat ion . By the sixth hour the germ tube had attained a length equal to that of the conidium. Ten hours after i so la t ion the f i r s t septum was observed and two hours la ter conidia formation started, apparently by fragmentation of the mycelium. These conidia contained abundant o i l droplets. These spores germinated as did 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 cy l indric 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 aerial hyphae has been observed to occur along the septa of the ce l l s resulting i n a chain of conidia set end on end. The length of the hyphal ce l l s , and hence the length of the result ing conidia appeared to be dependent on the culture medium used for growth. The second process of conidia formation i s more commonly known and i s described i n some detai l by Lehman (4). This method of formation involved the endogenous production of the spores which i s characterist ic 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 irm and entire - 9 -by the time the conidium was pushed out from the terminal opening of the conidiophore. The formation of the endoconidial wa l l was obviously not the resu l t of the long i tud ina 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) for the endoconidia of Thie lav ia bas icola (B & B r . ) . The wal l s of the new spores appeared to be generated by the c e l l protoplas t . There appeared to be two f a i r l y d i s t i n c t types of conidiophores. One type was morphologically indis t inguishable from the t y p i c a l hypha of the fungus. The other appeared to be more highly spec ia l ized for the production of endoconidia. The base of the c e l l was r e l a t i v e l y large and tapered gradually 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 previously described conidiophore. The l a t t e r type of conidiophore appeared to be a more p r o l i f i c producer of con-i d i a . 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 ive spore produced endogenously from w i t h i n the specia l ized mycelium and also 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 abs t r i c t ed . These spores apparently contained abundant food reserves and were observed to be produced not only t e rmina l ly at the end of the conidiophore, but a lso i n in te rca la ry posi t ions along hyphal strands. As a resu l t of these observations, these spores are r e -ferred to throughout t h i s paper as chlamydospores. The f i r s t spore formed i n a chain was observed to possess two d i s t i n c t w a l l s . Harter and Weimer (19) reported that these spores germinated r a r e ly . 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 did produce other chlomydospores by budding (Plated ). There has been no reference i n the l i t erature reviewed indicating this type of spore formation i n Ceratostomella. The end result was a number of spores eet end on end similar to those produced i n an endogenous manner. The mature pear-shaped spores were about 15/± to 19y long and 9jJ- to 11 y. wide. The mycelia producing these spores were about 6jj. i n diameter. In no case -was; the formation of chlomydospores observed on the surface of the medium. A l l chlomydospore formation appeared to 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 after 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 . When touched with the point of a needle the mass disintegrated. 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 irm globular body was quite apparent under the binocular microscope. The beak was the las t part of the f ru i t ing body to be formed. By the end of the fourth day the black globose and beaked fru i t ing 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 ru i t ing body. Microscopic examination of sections of the fru i t ing body at various stages of i t s development f a i l e d , at f i r s t , to give evidence of a sc i . 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 asci and perithecial develop-ment occur simultaneously and that the asci 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 perithecial cavity. The gelatinous matrix i n which the ascospores were exuded probably originated from the disintegrated asci walls and from the internal wall of the perithecium. The one-celled ascoperes occurred In hundreds i n these gelatinous exudations at the t i p of the perithecia. These masses were whitish-opaque at f i r s t but about fourteen days later they turned a smoky-yellow color . At this stage, ascospores, while s t i l l attached to the t i p of the perithecia, germinated producing slender hyphae which produced hyaline conidia as described before. The globose portion of the;perithecia had a diameter of lCO)i to 120JUL. 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 perithecia proved more successful. The perithecia were picked out from the cultures, fixed and k i l l e d i n an alcohol/acetic acid mixture for 20 minutes and then d irect ly in f i l t ra ted and imbedded i n paraffin wax. Hand sections were made and by this method, two intact asc i containing ascospores i n the perithecial cavity were obtained i n separate sections. The asc i were irregular ly sack-shaped and while the number of ascospores could not be clearly distinguished, four - 12 -seemed to be apparent i n each case. In cultures 12 days old a dist inct acetate-like odor of some apparently organic substance was discernible . The odor increased i n intensity u n t i l the colony stopped growth about three weeks la ter when the margin of the plate was reached. - 13 -A CYTOLQGICAL STUDY The object of th i s study i s to observe the nuclear structure and mode of d iv i s ion i n the resting and germinating conidia of the fungus. Although the germination of a spore appears to be a simple physical phenomenon, there are many small but essential ly constant cytoplasmic differences i n the germination of various fungus spores. It 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 half going to the growing point of the tube and the other half remaining behind. It i s also known that the time at which the f i r s t nuclear d iv i s ion occurs may vary. Some nuclei 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 iv is ion of the nucleus i n many fungi i s dbill an open question. Some workers state that the div is ion i s bas ical ly the same as that which occurs i n the ce l l s of the higher plants, involving the accepted theories of mitosis and meiosis. Robinow and other workers (20, 2L) are of the opinion that the nuclei of some of these lower forms do not undergo any organized d iv i s ion but divide by a more or less random pull ing apart of chromatin material which results i n two nucle i . Materials and Methods Using the method described on page 40 for studies on the induction of mutant strains, conidia were prepared for staining after growth had been in i t ia ted on dialyzing membrane. It was found that after four and a half hours at 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 ix ing conidia on the membranes at intervals during - 14 -the third to sixth hour after planting, a suitable range of various stages of germination was obtained. The fixative used was made up of 3 parts 95$ ethyl alcohol and 1 part glacial acetic acid. The membrane squares were placed directly into the fixative. The material was k i l l e d and fixed for a period of 10 minutes. Throughout this staining period small stenders especially constructed for staining procedures using microscopic cover slips were used. The membranes were then placed i n 10% ethyl alcohol for 10 minutes and then hydrolyzed i n IN hydrochloric acid at 60°C for 10 to 13 minutes. After washing in d i s t i l l e d water for about 10 minutes, the membranes were stained for 1 hour i n buffered Giemsa (buffered at pH 6.2). The membranes were washed in tap water before mounting i n water for immediate observation. Observations The resting conidium had an average length of 16>1 and an average width of 6jJL. The conidium usually contained two large o i l droplets at opposite ends and abundant cytoplasmic material containing many highly refractive bodies. The conidia were uninucleate with the nucleus centrally located. The resting nucleus stained blue as a dark crescent-shaped homogenous mass with a definite light 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 or-ganized reticulate system of chromatin material. Definite strands could be identified. The exact number, however, could not be definitely - 15 -ascertained because of the ret iculate nature of the mass. Seemingly the number of these strands vary between three and s ix , appearing as irregular l inear bodies in diverse shapes of Vs. The germ-tube i n i t i a l appeared about four to five hours after planting. More than one apparent germ-tube i n i t i a l was observed on conidia but i n no case did more than one germ-tube develop. The germ-tube i n i t i a l rarely appeared central ly , 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 this part of the c e l l wal l . The f i r s t nuclear d iv i s ion 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 position i n the conidium towards the area of the germ tube i n i t i a l . At th is site there appeared to be a mechanical separation of the diffuse reticulate material with the formation of a narrow isthmus across the point at which the germ tube leaves the conidium. After the d iv is ion, one-half of the material migrated to the t i p of the germ tube and the remaining half moved back to i t s or ig inal central location i n the conidium. The entire structure i s f i l l e d with cytoplasmic material. The second nuclear d iv i s ion was unique since i t was not the nucleus at the t i p which divided but, by a process similar to the f i r s t nuclear d iv i s ion , the parent ret iculate 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 or ig inal posit ion, while the other took i t s posit ion behind the nucleus at the t i p . Thus far the germ tube was coenocytic, containing two nuclei and no cross walls. - 16 -At about the time of the t h i r d nuclear d iv i s ion , a ce l l wall was observed between the two first-formed nuclei i n the germ tube. Five such divis ions, involving the parent nucleus i n the formation of new nuclei for the elongating germ tube, were noted before a d iv is ion of the nucleus at the t ip of the germ tube was observed.!Plate III) . Discussion No definite mitotic figures were observed i n any of the nuclear divis ions. The organization of chromatin from nuclear material appeared to be similar to that which occurs i n the prophase i n mitosis i n the cel ls of higher plants. The formation of new nuclei seemed to be a random pull ing apart of the chromatin material which resulted f i n a l l y inf .•> two nucle i . The l i g h t area interpreted as representing a central body was seen only i n the resting nuclei and was not observed at the time of the organization of the nucleus into chromatin material, nor during d iv i s ion . Various techniques involving the use of methyl alcohol and formalin for the f ixat ion and k i l l i n g processes resulted i n great d i s tor-t ion of the spores and induced the formation of ar t i facts i n the cytoplasm. The ethyl alcohol and g lac ia l acetic acid mixture which was f i n a l l y used appeared to be a milder fixative and caused l i t t l e d is tort ion. 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 divisions might have been due to energy stored up in the conidia and that the capacity for such divisions was retained u n t i l the new c e l l could obtain those nutrients which allow the d iv is ion 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 perithecia 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 certain nutrient media which lacked a vitamin or amino acid which the 'wild* type was able to synthesize but which the mutant was unable to produce. Fries (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 for growth and sporulation. Methods and Materials In this 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 original 'wild Table 1 The Effect 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. (Average of 4 plates/treatment) medium : : CfB 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 similar treatments and the average colony diameter i n four replicates was used as the growth criterion. Observations Isolate B. In treatment 1 some zonation was present i n the cultures but this was not very distinct. The mycelia were very irregular i n dimensions and direction of growth, feeble i n appearance and septation and c e l l size were variable. No chlamydospores were present but conidia were plentiful on the surface. Neither perithecia nor ascospores were observed. In treatment 2 the cultures were similar to 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 similar to those i n treatment 2. With the maximum amount of thiamine hydrochloride, (50 mgm. Th HCl/25 cc. plate) the mycelium was s t i l l irce;gular and feeble. The c e l l s , however, contained more o i l droplets than i n the f i r s t treatment. Chlamydospores were present but they were few and relatively small. Isolate 4x. With the lowest concentration of thiamine hydrochloride, mycelial growth was very sparse and the mycelium was very fr a g i l e , forming chains of conidia of irregular sizes. This occurred especially on the periphery of the colony* Septation of the mycelium was variable, resulting i n c e l l s of various sizes. The cultures were rich with perithecial i n i t i a l s but no mature perithecia 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 also contained more perithecial i n i t i a l s . The cultures were more heavily pigmented and the mycelial septation was very inconstant. The ce l l s varied in 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 . Mycelium was present i n the medium but was very sparse. 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 conidia, which were produced i n an endogenous manner, were observed i n the medium. In treatment 3 there was only a faint pigmentation toward the centre and abundant surface conidia were formed, apparently by fragmentation of the aer ia l mycelium. Under the conditions of this 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 start centrally i n the mycelium which was submerged i n the medium i n the cultures of Isolate 4x. The aer ia l mycelium was l ight colored. The chia>mydospores were produced i n the medium and were dark olive-green i n color. - 20 -THE EFFECT OF COLCHICINE ON THE GROWTH AND SPORULATION OF fSTRAINS* OF OPHIOSTOMA FIMBRIATUM The use of colchicine for the production of polyploid mutants i n plants has attained widespread acclaim, especially 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 ibres . 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 regularly carries the chromosomes to their respective poles. Hence the spindle f ibre i s the substrate where the alkaloid acts. 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 ibre and attending mechanisms. Some workers (Ostagren G. (27), and E i g s t i and Dustin (25)) consider that the fundamental relationship between the drug and the spindle f ibre i s a quantitative intermolecular reaction, 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 ter the morphological character and nuclear apparatus of the c e l l s . Levan (28), among many other workers, has successfully demonstrated the remarkable act ion of the drug. Treated ce l l s have the capacity to recover and segregate a bipolar 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 ce l l s treated with the drug. Changes comparable to those produced by X-rays have not been found. Only mutation involving the doubling of chromosomes have been known to occur. As a result i t i s incorrect to c lass i fy the drug as a true mutagen. From a review of the l i terature the action of colchicine - 21 -on the fungi appears to be very variable. Vanderwalle (31) found that the drug hindered the production of conidia i n Diaporthe perniciosa. Blakeslee (32), working with several species of fungi, did not observe any change due to the action of the a lka lo id . Several workers (Levan (33)» Beams (34), Laur (35), Vandendries (36)) fa i l ed to produce any changes i n cultures of Saxharomyces cerevisieae. Sinto and Yuasa (37) were able to produce cytological changes i n this yeast while E i g s t i andMs associates obtained enlarged cel ls by colchicine treatment. Grace (38) observed inhibi t ion while Richards (39) observed stimulation i n the production of yeast c e l l s . E igs t i and Dustin stated that changes i n the sizes of cel ls within a culture and direct action upon the growing organisms indicate that the drug has some influence upon the growth processes related to increase i n s ize. These changes are not transmitted to 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$ solution. Hindmarsh (41) used a 0.1$ solution to prevent forma-t ion and for the destruction of the spindle mechanisms .in; onion root t i p ce l l s . Nishiyama (42) used a 0.05$ solution to produce pentaploid hybrids i n oats. Walzel (43) used a 0.01$ solution to show inhib i t ion of seedling elongation, while Alcaraz and his associates (44) used 0.2 and 0.5$ solutions to produce tetraploid tobacco plants. Mader (45) obtained ce l lu lar 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 colchicine, ranging from 0.01$ to 2$. - 22 -Materials and Methods In a l l treatments potato dextrose agar was used as the basal medium. Five ninety m i l l i l i t r e portions of medium were s ter i l i zed by autodaving i n the usual manner. At the same time five test tubes, each containing 10 ml. d i s t i l l e d water and 0.5 gnu, 1.0 gm., 1.5 gm. and 2.0 gm. colchicine respectively (the 5th tube was used as a control) were s t er i l i zed . When the medium was cooled to about 50°C the various con-centrations of colchicine were added to the flasks of medium to make four treatments containing 0.5$, 1.0$, 1.5$ and 2$ colchicine. The medium i n the f i f t h f lask was used as a control . The flasks were then s t i rred to ensure the proper mixing of the medium with the added colchicine solution. From each flask four plates were poured. This process was repeated for each strain used i n the experiment. On each plate one planting was made central ly. The plates were incubated at 27°C. Observations were made from the tenth to fifteenth day after planting. Observations Table 2 The Effect of Colchicine on the Growth of Ophiostoma fimbriatum (Cultures 16 days o ld , 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 Cf .B . In treatment 1 and 2 the size and the septation of the mycelium were very irregular . The mycelium was indist inct and i n many cases collapsed. No chlamydospores were observed i n these cultures but there were abundant conidia on the surface of the medium. Zonation and banding i n a l l cultures were prominent. In treatment 3 the zonation was s t i l l manifest and the mycelium was irregular 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 en t i fu 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. Numerous conidia germinated to produce similar irregular 'buds'. These bodies varied from pear-shaped to club-shaped structures and many were detached and strewn on the surface of the culture. Some conidia were found to be dumb-bell shaped. 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 irregular , 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. Cultures of th i s isolate i n treatments 1 and 2 were s imilar. Large quantities of conidia were formed by fragmentation of the fragi le surface mycelium. The conidia were generally irregular . Chiamydospores were very numerous and were produced i n terminal intercalary and la t era l positions on the vegetative mycelium (Plate IV). Cultures i n treatments 3 and 4 were similar and noteworthy by - 24 -the fact that none of these produced chlomydospores. Conidia were produced i n large quantities in various sizes and shapes. Some were small and almost spherical, others were much more elongated and cy l indr ica l i n form. Isolate 2x. Cultures i n the f i r s t two treatments did not produce chlomydospores. Conidia, originating from inside the mycelium (endoconidia), were formed i n f a i r abundance i n the medium. Aer ia l mycelium was absent, since apparently a l l had been u t i l i z ed 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 cultures. The only apparent difference between the zones of banding i n the Cf.B isolate was the re lat ive abundance of mycelium. These cultures demonstrated certain pecul iar i t ies i n the morphology and germination of conidia. The cultures of isolate 4x were most p r o l i f i c i n growth. These cultures, apart from portraying conidial abnormalities, also exhibited idiosyncrasies i n the formation of ehlomydospore. Isolate 2x did not produce chlomydospores i n any treatment. The colchicine appeared to have affected the pigment system of this s tra in in some way. It i s to be noted i n particular that neither perithecia nor ascospores were borne i n any culture. The control cultures of Isolate B produced both types of asexual spores and perithecia yielding ascospores a l l i n the typica 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 isolate produced abundant conidia but yielded neither chlomydospores nor perithecia with ascospores. The control cultures of Isolate 4x were similar to those described i n the original isolat ions. Apart from the morphological changes and peculiar sporulation characteristics i n the cultures, the growth rate of a l l isolates i n the control cultures were greater than the same isolates 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 variety of plants to induce morphological and cytological variations. The action of camphor on plant ce l l s appeals to be a complex one, for Deysson (46) i n France obtained very diverse results , using different concentrations of the chemical on onion root t ip s . At a concentration of 0.02$ to 0.03$ there were no abnormalities i n c e l l size but there were cytological aberrations. At much lower concentrations (0.015$ to 0.012$) such enlargements were induced i n the area of c e l l elongation. Won (47) developed 'giant* ce l l s i n bacteria by incorporating from 25 mg. to 55 mg. of camphor i n 100 ml. blood agar. It i s his opinion that the rapid penetration of camphor into the bacterial c e l l was an important factor i n the production of 'giant' veriants. Subramanian and Roa (48) have indicated from their experiments that camphor had induced gene mutations i n several directions i n yeasts. Kostoff (49) found that a saturated solution of camphor inhibited the growth of mycelium of Penici l l ium sp. growing on honey agar. This treatment apparently encouraged branching, some of the branches being thicker and occasionally forming whole chains of giant conidia. Camphor i n large doses over a prolonged period prevented the formation of conidia. He noted that the giant conidia, when isolated, germinated and maintained the production of giant conidia through several transfers. Kostoff thinks that the gigantism was probably due to the doubling of the chromatin material. - 27 -Materials and Methods Comparatively high concentrations of camphor were used in these experiments to ascertain i t s effect on the test organisms. Di f f i cu l ty was encountered in dissolving more than 0.2 gms. of camphor in s ter i l e water, even by autoclaving. The camphor was dissolved and autoclaved separately and la ter added to double strength potato dextrose agar. Medium containing 0.2$ and 0.4$ camphor constituted treatment 1 and 2, respectively. Treatment 4 was made up of plates containing no camphor and was employed as the control . Four plates with a central planting of Isolate Cf.B were used in each treatment. Observations Table 3 The Effect of Camphor on the Growth and Sporulation of Ophiostoma fimbriatum ( A l l cultures were incubated for 22 days at 27°C.  Treatment Camphor Diameter Sporulation  gms/100 ml. of growth Ascospores Aer ia l Endo- Chlamydospores 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 in re lat ive ly small numbers. The perithecia were found to be aggregated around the central core and appeared sporadically, and much less 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 cultures. The y ie ld of perithecia and ascospores were accelerated i n these cultures. Copious amounts of conidia were produced in the medium i n an endogenous manner. The hyaline conidia, both aerial 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 similar to those i n the previous treatment, bearing regular, vigorous mycelium. The production of pigment and sexual spores was greatly increased. Perithecia were evenly distributed throughout the surface of the medium. Both the aer ia l and endoconidia were p lent i fu l and although there were much less chlomydospores than i n the control plates these cultures contained more of these spores than those growing on the medium containing less 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 addition of camphor to the medium and an increasing amount of camphor appears to increase the vigour and growth of the cultures. Di f f icul ty was encountered i n dissolving more than 0.2 gms. of the camphor i n the medium even by autoclaving at 215° and 15 l b . pressure. Camphor seemed to bring about a greater production of perithecia and ascospores and a smaller amount of chlomydospores. The control plates contained more chlomydospores than either of the camphor plates. The hyaline conidia were abundant i n a l l cultures. These appeared to be the type of spore most easi ly formed by t h i s fungus. The formation of this spore i n the medium was re la t ive ly rare. However, i n both camphor treatments the ir 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 culture, were amply produced but the method of production appeared not to be from inside the mycelium but by frag-mentation of the aerial 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 fungi . In th 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 nitrate 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 control . 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 d i s t i l l e d water before the addition of the other components of the medium. The complete media were then s t er i l i zed by autoclaving. Four plates were formed from each treatment l o t and planted centrally with Isolate Cf .B . The plates were incubated at 27°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  ( A l l cultures grew on an equivalent of 0.76% nitrogen at 27^6 for 16 days) Treatment Nitrogenous Amount Diameter Sporulation • compound (gm) of growth Ascospores Aer ia l Endo- Chlamydospores (cm)* conidia conidia 1 Asparagine 2 5.0 0 X 0 XXXX 2 Potassium 2.7 1.7 0 xxxx X 0 nitrate 3 Ammonium 1.9 0.6 0 XX XX XX sulphate 4 Urea 0.8 — no growth 5 Control — 4.2 XX XXXX XX XX *Average of four plates with numerous dark-black spots indispersed throughout the culture. The upper surface contained abundant a e r i a l mycelium and coindia. There was a widespread production of chlamydospores in the cultures. The mycelium was heavily pigmented and was re lat ive ly vigorous in 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 in places. Neither perithecia nor ascospores were observed in any cultures. Treatment 2 with potassium nitrate produced characteristic changes in 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 in Treatment 1, and did not appear to be pigmented. The hyaline cy l indric conidia were produced in copious quantities but^neither chlamydospores nor preithecia were - 32 -produced. Table 4 shows that growth i n these plates was much less than i n the previous treatment. In Treatment 3, ammonium sulphate was used as the source of nitrogen. The cultures here had a very limited 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 li g h t olive-green with a similar peripheral area. In general, the colonies were raised and were of a leathery consistency. Mycelial growth was irregular and the mycelium was very fragile. Conidia were few and the chlamydospores, though present, were not plentiful and a l l possessed relatively thin walls. No sexuality was observed i n the cultures. The treatment which contained urea hindered a l l growth of the fungus. Because of th i s fact and since the organism grew well on the control plates, i t was thought that either 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 toxic 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 to the basal medium before and after s t e r i l i z a t i o n when the medium had cooled to 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 irregular. The central areas (about 0.6 cm. diameter) were raised and light brown i n color. They consisted of weak, irregular, aerial mycelium with abundant o i l droplets, conidia of variable shaped and relatively 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 ter i l i za t ion of Urea on the Growth of Ophiostoma fimbriatum ( A l l cultures were grown at 27°C for 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 er i l i za t ion into compounds that were toxic or compounds rendered toxic by the condition involved i n the study to the growth of the organism. The unsteri l ized 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 analytical study of soi ls for micronutrients, used Aspergillus niger as a microbiological assay for copper. He found that when a nutrient solution was purif ied from copper by charcoal treatment (shaking with amorphous charcoal) Aspergillus developed only s ter i le white mycelium. The addition of ascending amounts of copper to the purif ied medium induced sporulation with increasing pigmentation of the spores unt 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 catalyst. In a second series of experiments the copper impurity of the nutrient was removed by recrysta l l i z ing the sa l t s . On a medium prepared from these salts and provided with an adequate amount of copper, the growth of Aspergillus was very poor. He thought that the nutrient medium purif ied with charcoal, though free from copper, contained some other substance which was removed i n the process of recrysta l l i zat ion of the nutrient salts and the sugar. Since copper was shown to influence the formation of pigments i n Aspergillus niger by Mulder (51) and Sakamura (52), i t was decided to test the effect of copper-deficient nutrient on the growth of th is fungus (0. fimbriatum). Methods and Materials The method of puri f icat ion involved, i n a modified manner, the processes used by Mulder and Sakamura. The culture medium employed com-prised the following ingredients: - 35 -Dextrose 20 gms. Sodium nitrate 2 gms. Potassium dihydrogen phosphate 2 gms. Sodium chloride 5 gms. Magnesium sulphate 0.5 gms. D i s t i l l e d water 1000 ccs. Amorphous* charcoal was shaken intermittently, over a period of 30 minutes, with ample sulphuric ac id . The acid 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 60°C, for 3 hours. This charcoal was added to the l i q u i d medium i n excess (about 150 gms/500 cc) . This mixture was shaken vigorously intermittently over a period of 2 hours, and then f i l t e red through f i l t e r paper, treated with dilute sulphuric acid, washed with double d i s t i l l e d water and dried i n the oven. For further purif icat ion calcium phosphate was used. This phosphate was f i r s t purif ied by shaking i n double d i s t i l l e d water (50 gm/litre) over a period of 4 hours during which the water was changed four times. The mixture was f i l t e red as described before. The purif ied calcium phosphate was added to the culture solution (0.5 gm/lOO cc.) and, using sodium hydroxide, the reaction of the solution was adjusted to pH 5.5. This mixture was also shaken periodical ly for 2 hours and f i l t ered as before. The glassware to be used was a l l f i l l e d with 1% solution of purif ied calcium phosphate and autoclaved for 20 minutes at 15 lb 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 puri f icat ion were not observed since i t * From glass s t i l l and receiver. - 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 cc. of the purif ied medium were added to each of s ix 'copper free 1 250 cc. Erlenmeyer f lasks. To another 300 cc. of purif ied 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 Aspergi l lus) . This medium was equally distributed into s ix more flasks as before. A l l flasks were planted with mycelial fragments from th ird generation cultures of Isolate B grown successively on 'purif ied medium'. The inoculum con-tained no spores and consisted ent ire ly of hyaline mycelium with a cottony appearance. As a control, six f lasks containing 50 cc. of a modified Czapek's solution were also planted with the 'copper deficient' 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 at the end of 15 days, the growth i n each flask was f i l t e red through individual ly weighed f i l t e r papers which were ovenj»dried i n desiccators at 30°C. for 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 cultural differences i n the various media used. The mycelium i n the cultures grown i n the Cu-deficient medium contained no observable amount of pigmentation. The mycelium was re lat ive ly - 37 -very sparse and irregular i n c e l l size and i n the width of the hyphae. The cultures were completely s t er i l e , neither sexual nor asexual spores being observed. The cultures grown i n the 'deficient medium', to which copper was added, showed marked pigmentation. The older mycelium i n the centre of the culture was olive-green i n color. The mycelial t ips were hyaline and cy l indr ica l conidia were endogenously produced. The mycelium was definitely more vigorous i n appearance, with thicker walls and o i l globules i n most of the ce l l s . The cultures grown in the modified Czapek's solution were darker olive-green i n appearance, indicating 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 is t inct septation and although the hyphae t ips were much l ighter 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 th i s medium, and conidia and dark grey-green, pear-shaped chlamydospores were present. The hyaline conidia outnumbered the dark-celled chlamydospores by about 50:1. The chlamydospores were produced mainly i n the older central area of the culture. No mature perithecia were observed but numerous peri thecial i n i t i a l s (as identif ied 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 this stage fa i l ed to reveal any asci 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 Effect of Copper-Deficient Medium on the Growth of Ophiostoma fimbriatum Culture No. Weight i n grams of 10 day old cultures 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 Average 0.045 0.102 .22 Conclusions From the weights of the cultures grown i n the various media, i t i s evident that copper deficiency affects the physiology of growth i n th is fungus. A l l cultures grown i n the 'copper deficient' medium produced less growth than the other cultures. It 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 for these deficiencies has been treated ear l i er . It i s thought that the greater growth under Treatment 3 i s due - 39 -to the fact that the purif icat ion process i n Treatment 2 probably removed other necessary metals from the solution. The medium under Treatment 3, being unpurified, contained enough micronutrients for 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 direct or indirect effect on perithecial formation. Fries (53) observed that a mutant s train of 0. multiannulatum. which could not synthesize i t s own tyrosine, remained without pigment. From work with strains 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 perithecia. From these observations i t appears that copper and tyrosine metabolism may have some correlation with perithecial formation. It i s believed that peri thecia l formation under Treatment 3 was inhibited because of two probable reasons: 1. Unsuitable substrate 2. Inadequate aeration Using a similar medium with the addition of agar, this fungus produced perithecia i n a 1 G day old culture. It i s thought, therefore, that the l i q u i d medium provided osmotic conditions which were unsuitable for perithecial formation. Also, perithecia have been observed to occur superf ic ia l ly or only s l ight ly immersed i n the substrate whether the fungus i s growing on a r t i f i c i a l media or naturally on a susceptible host. This tends to indicate that aeration may play some role i n the formation of perithecia i n this 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 inhibi t ing perithecial formation in Treatment 3. - 40 -From this and other studies i t i s noted that hyaline conidia are the spores most easi ly formed i n culture followed by chlamydospores, and f i n a l l y by the sexual stage as nutrit ional conditions improve. - 41 -THE INDUCTION OF MUTATIONS BY X-RAYS AND ULTRA-VIOLET RAYS From the work of Timofeeff-Ressovsky (54), Sinnott and his colleagues (55) concluded that the frequency of mutation induced by X-rays i s d irect ly proportional to the amount of radiation. 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 . Also, a certain amount of X-rays has the same effect regardless of whether i t is administered to the organism in a short time from a powerful source or over a longer period from a weak source. If 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 effects of X-radiation are cumulative. Mutagenic changes brought about i n ce l l s treated with X-radiation 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 this theory a mutation i s caused by a single ionization or atomic excitation within a certain 'target*. It i s assumed that a single 'hit* anywhere i n the target w i l l cause a gene change. These changes are very loca l i zed . Chromosomal changes are usually more crude mechanical changes involving breakage of the chromosomes and resulting i n deficiencies, duplications, translocations and inversions. Non-disjunction and loss of chromosomes are also frequent i n ce l l s treated with X-rays. - 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 action 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 rejoin other broken chromosomes and thus produce aberrations. I f , however, the end rejoins the or ig inal 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 rradiat ion resulted from ionizations within the chromosome i t s e l f . Wyss and his colleagues (58) furthered this view by inducing mutations i n Staphylococcus aureus by chemical treatment of the substrate with hydrogen peroxide. The object of this experiment i s to obtain mutant strains of this fungus which di f fer from one another i n gross morphologic character-i s t i c s , such as colony color, 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 irradiat ion of spores, several small squares (about 1 cm. sq.) of dialyzing membrane which had been s ter i l i zed by boi l ing for 15 minutes i n a Petr i dish, were placed contrally on a s ter i le - 43 -Petr i dish containing about 25 cc. P.D.A. as shown i n Figure 1. Using a s ter i le wire loop, a suspension of spores -was; gently streaked over the membrane squares, care being taken to use a re lat ive ly high d i lut ion of the spores. The spores were irradiated immediately or allowed to germinate and treated 6 to 8 hours after planting. After treatment each membrane square was transferred aseptical ly and planted centrally on a s ter i le P.D.A. plate and the spores allowed to grow. For culture treatment, a (2 mm. diameter) disc of inoculum was planted centrally on a s ter i le 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 Pe tr i dish i n posit ion, 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 for sectoring or abnormal growth and from such areas isolations were made. Later , these cultures were examined and compared with the or ig inal culture for morphological difference. The machine used for irradiat ion was a superficial X-ray unit . The plates were treated individual ly and were placed on a wax block to ensure the true dosage at a F .5 .D. of 15.9 cm. The machine was run at 90 K.V. and 10 milliamperes constantly. In some treatments a g mm. aluminum f i l t e r was used. This effectively reduced the rate of i rradiat ion but screened out soft radiation. In some cases no f i l t e r was used. From a review of the l i t era ture , iib appears that there i s a wide variation in the amount of radiation required to induce mutation even between closely related organisms. - 44 -Emmerling (59) treated the chromosomes of Zea mays with X-radiation and administered doses ranging from 400 r to 1000 r . Deschuer and Sparrow (60) irradiated 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) irradiated ascospores of Ophiostoma multiannulatum with a Coolidge tube at 50 Ki lo volts and 2-3 milliamperes for 100 minutes at a distance of 6-12 cm. and obtained mutant strains which were unable to synthesize certain chemicals necessary for growth. As a result i t was decided that a wide range of doses would be administered, starting at 50 r up to a maximum of 60,000 r . Details of rate and duration of doses are shown i n the following table. Radiation Experiment Voltage - 90 K.V. F . S . D . - 15 cm. Current - 10 milliamperes Rate - 115 rontgens per minute F i l t e r - 2 A l (H.V.L.2 .3 , Al ) A l l treatments were continuous Dosage P.D.A. plates Duration of application (rontgens) No. (duplicated) 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 original culture obtained from Dr. Martin in Louisiana was label led isolate C f . B . This was a dark olive-green colony which produced perithecia, ascospores, chlamydospores and conidia. At f i r s t , ascospores and perithecia were produced centrally i n the oldest part of the culture, and la ter perithecia were found scattered randomly throughout the colony. The perithecia had a diameter of about 100p. The neck was about 200JJ long and fimbriate at the t i p . The ascospores had a diameter of approximately 3)i. The hyaline, thin-walled conidia were produced aer ia l ly and endogenously i n f a i r abundance and measured about 12jax 6jjtin length. The chlamydospores were produced i n the medium, at f i r s t endogenously and later by a process of budding. They were com-paratively heavy-walled, olive-green i n color, 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. The diameter of the mycelium was about 6 jJ . Seventy day old colonies growing at room temperature ( 2 0 - 2 4 ° C . ) had an average diameter of 6.6 cm. These colonies were dark oEve grey-green and not uniform i n appearance. There was no banding i n the cultures and perithecia and ascospore production were abundant. 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 cyl indric 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 ight ly slower (2.2 cm.) during the i n i t i a l 5 days and - 46 -2.7 cm. for the las t 10 days (See table 7). Isolate IX - This "mutant" was isolated from plate 7. The or ig inal isolate did not produce perithecia ;or ascospores, nor did i t produce any cy l indr ic conidia. Chlamydospores, however, were produced and the mycelium was delicate and uniform. Several isolations from fanfshaped sectors were made on potato dextrose agar plates (four plantings per plate) . After about four days growth the cultures were examined, and then from those which maintained their cultural differences from the or ig ina l , transfers were made to four potato dextrose agar plates, making one planting per plate central ly. After 6 days growing at 27°C. the cultures were examined. The average diameter of the cultures was 3 cm. The cultures were completely s t er i l e , with slender irregular mycelium containing abundant o i l droplets. The cultures were regular i n outline and l ight olive-green i n color. After growing for 70 days at room temperature the colony attained an average diameter of 5.6 cm., with the following cultural characteristics: The colony was dark olive-green with definite banding. There was a raised white area i n the centre, 2 cm. in diameter. On the underside there was a dark central core (2 cm. diameter), corresponding to the l ight raised area above, surrounded by concentric zones of alternating l i ght and dark color. This colony produced no surface conidia, but chlamydospores were p lent i fu l in the medium. Perithecia were absent and no perithecial i n i t i a l s were observed i n the four platew examined. Serial transfers were made and the f i f t h generation colony, 21 days old, was examined. The characteristics remained true as described before except that the production of chlamydospores was greatly 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. Isolate No. Plate No. 5th day 7th day 25th day C. f . B. 1 no growth no growth 0.6 2 ti it 0.3 IX 1 0.2 0.2 0.2 2 0.2 0.2 0.3 2X 1 0.3 0.3 0.3 2 0.4 0.4 0.4 4X 1 0.5 0.5 0.5 2 0.5 0.5 0.5 5X 1 0.5 0.5 0.5 2 0.5 0.5 0.5 6X 1 0.5 0.5 0.5 2 0.5 0.6 0.6 7X 1 0.5 0.5 0.5 2 0.5 0.5 0.6 8X 1 0.4 0.5 0.5 2 0.5 0.5 0.6 9X 1 0.6 0.65 0.7 2 0.6 0.7 0.7 10X 1 0.5 0.5 0.6 2 0.5 0.5 0.5 - 48 -Table 8 Growth Rate of Isolates on Potato Dextrose Agar at 2 7 ° C. Diameter i n centimeters  Culture Plate Av. d i f f . Av. d i f f . Av. d i f f . No. No. 5th day i n growth 10th day i n growth 15th day i n growth C. f. B. 1 2 3 4 2.1 2.3 2.2 2.1 4.9 5.0 4.6 4.9 7.3 7.5 6.6 8.0 2.2 2.2 4.9 2.7 7.6 2.7 IX 1 2 3 4 2.2 2.2 2.3 2.2 • • ' 4.0 4.6 4.5 4.6 6.5 6.5 6.5 6.5 2.2 2.2 4.4 2.2 6.5 1.9 2X 1 2 3 4 2.3 2.2 2.3 2.2 3.5 4.5 5.0 3.3 6.2 7.3 6.0 2.25 2.3 4.07 1.8 6.5 2.4 4X 1 2 3 4 2.5 2.5 2.6 2.6 5.5 5.0 5.1 5.0 7.5 7.5 7.6 2.55 2.6 5.15 2.60 7.53 4.9 5X 1 2 3 4 2.1 2.0 2.1 2.1 5.5 6.5 6.5 6.5 2.1 2.1 6.5 6X 1 2.3 4.6 7.4 2 2.1 4.5 7.0 3 2.4 4.5 7.0 4 2.1 4.4 6.6 2.2 2.2 4.5 2.3 7.0 4.7 7X 1 1.8 3.6 6.2 2 1.6 3.5 5.7 3 1.7 3.3 6.0 ft - - -1.7 1.7 3.47 2.0 5.97 4.2 - 49 -Table 8 (Cont'd) Diameter i n centimeters Culture Plate Av. d i f f . Av. d i f f . Av. d i f f . No. No. 5th day i n growth 10th day i n growth 15th day i n growth 8X 1 1.7 3.3 5.5 2 1.7 3.5 6.0 3 1.8 3.5 6.0 4 1.8 3.4 5.9 1.75 1.75 3.42 1.67 5.85 4.18 2.6 5.0 7.5 2.8 5.3 7.5 2.6 4.4 7.5 2.5 5.2 7.5 2.62 2.62 4.79 2.35 7.5 2.53 10X 1 1.7 3.3 5.1 2 1.6 3.2 5.9 3 1.6 3.3 5.6 4 1.7 3.4 5.5 1.65 1.65 3.3 1.65 5.52 3.87 fungus grew to 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 slightly faster during the f i r s t 10 days than during the l a t t e r 5 days. This strain i s slightly slower growing than the original isolate B. From strain IX, three further isolations (lXa, lXb, and lXc) were made from sectors i n the colonies. lXa - This isolation 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 rest 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 cells - 50 -were very long. Neither perithecial initials nor conidia were observed, and only one chlamydospore was found. lXb - This isolate was obtained from a. IX colony growing at 27°C. on P.D.A. This sector was very d i s t inc t ly darker than the rest of the colony. The mycelium was more abundant than in l X a , varying from color--less , 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 cel ls were shorter and the c e l l walls thicker than i n l X a . This isolate was completely s t e r i l e , producing neither sexual nor asexual spores. The colony was slower growing than IX, attaining an average diameter of 2.2 cm. i n 6 days at 27°C. lXc - This isolate was made from a fam*shaped sector i n one of the IX cultures. It was similar to the or ig inal IX i n most respects except that the colony was much l ighter 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 faster than that of the or ig ina l . Peculiar anastomosing hyphae was observed i n mounts made from the culture. 2X - This isolate was obtained from a sector i n Plate 17. The orig inal culture was dark grey-green with a central abundance of perithecia and also a narrow (4 mm.) band containing perithecia at a distance of 2 cm. from the centre. The sector was much l ighter i n color and contained no perithecia. After 26 days growing at room temperature on P.D.A. the isolate attained a diameter of 4.7 cm. with a central dark olive-green core, 2.2 cm. i n diameter. The rest of the culture was a l ighter color, similar to the or ig inal sector. No perithecia nor chlamydospores were seen. Surface conidia and endogenous conidia i n the medium were abundant. - 51 -Seventy day old cultures growing under similar conditions had an average diameter of 6.5 cm. (average of 4 plates) . The cultures were l ight grey with no aer ia l mycelium but contained abundant grey-white surface conidia. No mature perithecia were observed, but a narrow (3 mm.) band of peri thecial i n i t i a l s were seen at a radius of 1.5 cm. from the centre. Another dist inct continuous dark band was found at a radius of 2.5 cm. from the centre, the mycelium i n t h i s area being a darker ferey-green with no perithecial i n i t i a l s . A sixth generation culture, 6 days old, grown on P.D.A. had a diameter of 3.5 cm. This culture contained numerous perithecia, no chlamydospores, and abundant surface conidia and conidia produced endogenously i n the medium. This culture suggests some reversion to the or ig inal C. fimbriata, isolate B. 4X - This isolate was obtained from a sector on Plate 17. The sector contained an increased number of perithecia and perithecial i n i t i a l s . A seventy day old culture grown on P.D.A. at room temperature produced no perithecia, but there were numerous perithecial 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. The colony had an average diameter of 6.3 cm. From this isolate a sub-isolate was made from a sector i n one of the cultures. This was label led 4Xa. 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 ght olive-green i n color i n the medium but the surface was much l ighter grey with a mealy consistency. No perithecia were observed. Conidia were more numerous than i n 4X and chlamydospores were p l en t i fu 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 perithecia nor perithecial i n i t i a l s i n the 4X cultures, whereas the 4Xa cultures contained a copious amount of peri thecial 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. No chlamydospores were observed i n any of these cultures. 9X - This isolate 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 rrad ia t ion , was grey-green i n color on the edge of the colony. Immediate examination showed that chlamydospores were plent i fu l and the endogenous production of chlamydospores and conidia was widely distributed. 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 isolate was definitely faster growing than the other i solates , and the cultures showed a very definite banding. These cultures produced no perithecia. Chlamydospores were f a i r l y numerous and conidia were few. The mycelium was hyaline, no pigmentation being observed. The mycelium was not very uniform i n width and did 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 isolate was made from a culture of C_. fimbriata B, which was not treated with X-rays. Sectoring was observed on the culture and - 53 -isolations were made. Simultaneous observation indicated the presence of a dumb-bell shaped bacterial contaminant widespread throughout the medium. This contaminant was eliminated by adding acetic acid to the medium to reduce the pH. The sector was brown-grey i n color, i n contrast to the grey-green of the parent culture. 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 (12/x - 14/A.). NO chlamydospores were observed and perithecia were absent. F i f th generation cultures growing on potato dextrose agar at 27°C. for 6 days had an average diameter of 3.3 cm. and the cultures were similar i n many respects to the or ig ina l . The colony did not maintain i t s brown-grey color but was not very l ight olive-green, much l ighter than the or ig ina l . The mycelium too, was less pigmented, almost colorless i n appearance, weak and irregular in growth. Conclusions From the tables i t can be seen that isolates B and 9X had about the same rate and amount of growth. At f i r s t , the average increase i n radia l growth was higher i n 9X ( 2 . 6 2 ) as compared to that of isolate B ( 2 . 2 ) . In the la ter stages, however, isolate B had a greater average increase i n growth. The rate of growth of isolate IX seemed to be f a i r l y constant. Isolate 9X grew the fastest of a l l the cultures i n the f i r s t five days. The rate of growth of isolate A increased with time unt i l the fifteenth day. In general, during the las t five days most of the isolates increased their 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 pollen grains (Deschner and Sparrow, 60) to over 50,000 r . for Baci l lus 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 this more drast ic treatment and to determine the amount of X-radiation that i s l e thal to the organism. Materials and Methods The medium employed here was modified Czapek's Agar, the same as that used earl ier i n this study. Conidia and ascospores i n a water suspension were spread out on strips of dialyzing membrane and placed centrally on the Petr i plate as dewcribed previously. The spores were irradiated one-half hour after being taken from the cultures and placed on the membranes. The superficial X-ray unit was used at 10 milliamperes and 90 Ki lovol 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 calculation of the doses compensation was made for the Pe tr i dish l i d which was l e f t i n position 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 plates, three 'mutants' appeared. These were observed microscopically, isolated and label led 55Xa, 55Xb, and 55Xc. - 55 -Dosage i n Duration of Treatment i n Treatment Rontgen Units Minutes 1 20,625 55 2 48,000 128 3* 50,000 90 4 (control) * A three day old culture treated without 0.5 mm. aluminum f i l t e r . Isolate 55Xa was completely s t er i l e , producing white a e r i a l mycelium and microscopically very similar to isolate IX obtained after treatment with 400 r . Isolate 55Xb appeared as a darker olive-green sector, as compared to the original C f . B , and contained numerous conidia, both on the surface of the medium and i n the medium. Neither chlamydospores nor perithecia were observed. The mycelium was vigorous and uniform. Isolate 55Xc produced perithecia, 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 isolates 55Xb and 55 Xc remained true to the change but reverted to the or ig ina l . Spores i n treatment 2 showed very low percentage germination even after 23 hours. It was estimated that the percentage germination was below 1%. Percentage estimates were made from counts of the number of germinated and ungerminated spores i n twelve random and separate f ie lds under the low power (xlOO). No growth resulted from transfers made of germinated spores. - 56 -The 3 day old culture used in Treatment 3 was 2.5 cm. i n diameter and contained large quantities of conidia, butneither chlamydospores not perithecia were present. A high dosage was administered i n an attempt to estimate the l e tha l effect of radiation. The appearance of the culture after i rradiat ion was markedly different from that of the un-treated Cf.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 color. This area contained numerous conidia and perithecia with ascospores, especially at the centrej a few chlamydospores were present. The mycelium here appeared def ini te ly darker and more vigorous than that outside this locat ion. On the immediate periphery of this area there was a dist inct complete band (5 mm. wide) of perithecia bearing gelatinous masses of ascospores on the ir fimbriate t ips . Beyond this band, the colony consisted of dark.mycelium (but l ighter than that i n the central core) and more abundant but smaller perithecia i n scattered clusters. Chlamydospores were obviously more prevalent here than i n the central dark area. No 'mutants' have been observed from sub-cultures. Conclusions 1. Vegetative growth appears much more, resistant to irradiat ion than spores. 2. Apparently ident ical mutants seem to occur independently under different treatments. 3. Comparatively, this fungus i s very resistant to the l e tha l effect of X-radiation. - 57 -4. An approximate estimate of cultural changes show about 2$ mutations, about 1.5$ reversion and about 0.5$ remained true to change for 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 or ig ina l . 6. Continuous growth on the same medium (P.D.A.) appears to induce degeneration i n the isolates . 7. After 4 months 'mutant' isolates produce subsidiary "sports" spontaneously i n culture. Six such isolates were indis t ing-uishable morphologically from isolates already made. 8. As a result of th is i n s t a b i l i t y i n the cultures i t i s proposed that a l l isolates be termed 'mutants'. It has been found that i n mutation work the agar colony technique has the l imitat ion of permitting detection only of those colonies whose mutation i s reflected by conspicuous change in cultural or morph-ological appearances. Probably a far 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 culture, 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 for reversion of cultures i s s t i l l not clear. Steinberg and Thorn (62) suggested that some mutations, at l eas t , might be due to alterations i n c e l l protein (chromatin, enzymes etc.) of the fungus, and further suggested that reversion consisted of repair of chemical injury to c e l l protein. This suggestion lends i t s e l f to the speculation that the gene may be present and intact 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 strain of Neurospora sitophila grew normally on an acid medium with ammonium-nitrogen. - 59 -THE EFFECT OF ULTRA-VIOLET RAYS ON SPORES OF OPHIOSTOMA FIMBRIATUM Petr i plates containing s ter i le dialyzing membrane were prepared as described before. Conidia and ascospores, i n a dilute suspension were placed on the membrane sections and also d irect ly on the agar surface. These were treated i n a steri le 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 for 5» 10, 15, 20, 30, 40, 45 and 60 minutes. Observations The majority of spores, especially the conidia, 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 less than that on conidium germination at 15 minutes treatment. Ascospores did 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 after 40 minutes treatment but on transfer 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 isolate (16X) was obtained and this was made from a 5 minute-treated culture. So far , 16X appears to be identical to a previous isolate , 9X. - 60 -The Cellophane Membrane Method The cellophane membrane method of spore germination for mutation work has several advantages. F i r s t , i t f a c i l i t a t e s the transfer of germinated spores. Thus, by treating.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 poss ib i l i ty of growth and the appearance of mutants by reducing the competition i n growth with the parent 'wild type*. This method also fac i l i ta te s re lat ive ly easy cytological study of irradiated, germinated or ungerminated spores, or the cytology of the irradiated growing germ tube. On suitable media the germination can be followed d irec t ly , 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 ixing solutions, alcohols, stains, and even acid hydrolysis without much los s . By th is method germinating spores could be subjected to several different nutrient treatments merely by transferring the germinating spores on the membrane. Instead of several different irradiat ion treatments of smaller amounts of organisms th i s method provides for 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 so la t ion , espe c i a l l y for spores that are comparatively small and where germination i s necessary before i so lat ion . Several (1-2 dozen) small squares (1-2 mm^ ) - 61 -of s ter i le membrane paper are placed on an appropriate nutrient media, a suitable d i lut ion of the spores i s poured on the plate over the membrane sections. At the desired stage of germination a single spore i so lat ion can be made by removing a membrane section containing one spore and re-planting on another plate . - 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 isolates be subjected to a pathogenicity test to observe their relat ive degrees of virulence on the susceptible host t issues. 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 inches. Care was taken to retain the epidermal ' sk in 1 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 semi-upright position with the epidermis upwards. To each flask was added 10 cc. of water. The flasks were stoppered with cotton and s ter i l i zed i n the autoclave. After s t er i l i za t ion the tissues i n the flasks were 1 inoculated with 10 different isolates of the fungus, each block i n two flasks being iisnoculated with the same i so late . The xhnoculations were made by deposit-ing the .'..inoculum in a needle break i n the epidermis of the potato block to simulate natural jimoculations. The flasks were incubated at room temperature. Observations Both direct and microscopic observations were made to determine not only the pathogenicity but also the cultural characteristics of the - 63 -isolates growing on th is natural substrate. The infection on the skin caused by isolate IX, consisted of a few relat ive ly large raised postules. The aer ia l mycelium on these postules was white. The infection i n the internal tissue produced a grey coloration. The centre of the raised 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. Chlamydospores were present but were re lat ive ly very few. 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 isolate 4X appeared to be a very virulent isolate . Con-s istently , a l l four blocks i n the two flasks los t the ir or ig inal conformation and became a soggy grey mass. The mycelium was dark olive-green, vigorous and abundant. Copious amounts of conidia and chlamydospores were present and perithecia and ascospores were produced i n isolated areas over the tissues. The isolate 5X appeared to be one of the least virulent i solates . Conidia were extremely p l en t i fu l , especially on the surface. Chlamydospores were present but very scant. Perithecia and ascospores were also 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 isolate 6X. This appeared to be a re lat ive ly virulent s tra in . The mycelium was dark olive-green on the surface. 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 lent i fu l and the cultures were teeming with perithecia and ascospores. Isolate 7X was similar i n cultural characteristics to oX but was much less virulent . Isolate 8X was obviously the most virulent i so late . The fungus involved the entire epidermal and central tissues of the sections, bringing about complete destruction. Conidia and chlamydospores were p lent i fu l but perithecial production was scant. Large raised white lesions were produced by isolate 9X. These consisted of very good mycelial growth but l i t t l e penetration into the cort ica l tissues was observed. Conidia were i n great numbers. Chlamydo-spores, though p lent i fu l , were not fu l l y developed. The chlamydospores were pear-shaped but thinner-walled and smaller and contained much less pigment than the typica l dark grey-green heavy-walled spore of this type. The mycelium was abundant, slender, hyaline, containing no pigment. The dark color of the sections around the lesions appeared to be due to oxidation of the host t issues. No perfect stage was observed i n this culture. Both sexual and asexual spores were present i n abundance i n cultures os isolate 10X. The mycelium were heavily pigmented and appeared as a dark mass. Isolate 15X was not very virulent and was comparable to isolate - 65 -IX and 9X in virulence. Str ik ingly , no conidia were produced but a few chlamydospores were found i n the lesions. The mycelium was dark grey-green and vigorous. On one of the potato blocks a » s p o r t ' appeared as a raised white cottony mass. Isolations were made from this and observa-tions revealed that this portion of the culture was completely s ter i le and produced l i g h t , hyaline, slender mycelium i n contrast to the vigorous pigmented mycelium of the original culture. 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 visual comparison based not on the rate but rather the amount of destruction of sweet potato t issue. The lesions varied from local ized and restr icted 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 overall study, that the hyaline cyl indric conidia are the type of spores most easi ly produced i n culture. Chlamydospores and perithecia are more reluctantly formed and their 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 ight ly different from an ordinary mycelial strand. The terminal producing c e l l i s larger and tapered toward the t i p with a basal c e l l that appears bulbous. Several conidia are produced from a single conidiophore. The second method of conidium formation i s by the fragmentation of the aer ia l mycelium and was observed to occur on the - 66 -surface of the substrate. In most cultures what appeared to be surface mycelium broke into innumerable unicel lular 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 sites and also to the osmotic interrelationships between the medium and the fungus hypha. The other asexual spore i s referred to i n this study as a chlamydospore since i t has a re lat ive ly heavy wal l , contains stored food i n the form of o i l or fa t and was seen to be produced, not only l a t e r a l l y and terminally, but also from the intercalary regions of the mycelium (see p. 23). Although the majority of these spores are produced endo-genously, 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 terature reviewed, these spores are alluded to as conidia, 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 ce l l s seems to be correlated to the vigour of the cultures. It appears, a l so , to affect the degree of pathogenicity of the isolates . O i l globules accumulate i n cel ls of less pigmented hyphae. The pigment i n t h i fungus i s retained within the ce l l s and does not diffuse out into the medium. Cultures of isolate B grown i n the dark and i n l i ght showed no apparent difference i n the degree of pigmentation. Two isolates which do not produce perithecia and sexual spores were planted on the same plate. No sexuality resulted from the meeting of the hyphae, suggesting either the incompatibility of heterothallic strains - 67 -or the i n a b i l i t y of the single or mixed isolate cultures to produce sexual bodies under those conditions of growth. It i s also to be noted that •sports' s t i l l arise from the sixth and seventh generation sub-cultures of the or ig inal irradiated spores. There are, however, many reversions. Isolate B haw been found to be inhibited i n growth by a species of Penici l l ium. A P P E N 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 II - (a) Diagrammatic representation of the endogenous production of the hyaline conidia and (b) the helmet-shaped ascospores. - 69 -PLATE II a b o PLATE III. Stages in the division 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 thin areas of the c e l l wall. (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 tip.(V) Migration of ghromatin material. (VI) Second nuclear division. (VII) as i n (V). (VIII) Third nuclear division and the inception of the f i r s t septum. (IX) A later stage of (VIII), showing completed cross-walls and the f i r s t division of the t i p nucleus. PLATE IV, Photomicrographs showing abnormal and germination of the sexual spores of 6. fimbriatum treated with colchicine. (I) The formation of thin-walled, pear-shaped bodies l a t e r a l l y on the vegetative hypha. (II) A similar 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 in (II) but with further budding. (VII-IX.) Terminal, l a t e r a l and intercalary formation of chlamydospores. For further discription 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) Control. (3) Potassium ni trate . (4) Ammonium sulphate. A l l cultures 16 days o ld . - 72 -PLATE V PLATE VI. Small aquares of dialyzing membrane placed on media and ready for planting. (Ixa) A culture of this isolate showing a further fan-shaped 'mutant' sector. (l6) and (4) Comparison of the growth and color characteristics between isolates 16X and 4X. (Note isolate 16X on dark background). - 73 -PLATE VI PLATE VII. Cultures of 0. fimbrlatum growing on different 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 - R A Y T R E A T M E N T S H O W I N G T H E R E L A T I O N S H I P B E T W E E N D O S A G E IN R O N T G E N UNITS AND T I M E O F A D M I N I S T R A T I O N 2 4 6 8 10 12 14 16 T I M E IN M I N U T E S - 76 -T H E S U P P O S E D R O L E O F C O P P E R IN T H E F O R M A T I O N O F O X I D I S E D M E L A N I N P I G M E N T IN O. F IMBRIATUM H O r C H i C H - C O O H * i N H C H , C H - C O O H - C u * N H , T Y R O S I N E E N Z - C i A O , 1 Redox C H C O O H NH. E N Z - C i / I Redox J Q E N 2 -C H C O O H - C u * N H , I o C H C O O H N H , Polymerization I TYROSINASE -TYROSINE - C U P R O U S C O M P L E X TYROSINASE (REDUCED) TYROSINASE (OXIDISED) 3.4-DIHYDROXY PHENYL ALANINE (DORA) TYROSINASE-DOPAQUINONE C U P R O U S C O M P L E X DOPAQUINONE OXIDISED MELANIN - n -BIBLIOGRAPHY 1. Halsted, B.D. Some fungus diseases of the sweet potato. N . J . Agr. 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Subramanian, C.V. and S.K. Sreepathi Rao. Studies on the mutagenic action of chemical and physical agencies on yeasts. Proc. Indian Acad. S c i . Sec. B. 35: 1-27. 1952. 49. Kastoff, Dontcho. Gigantiz m p r i Penici l l ium ogitno poulchen. Izvest i ia na Kamarata na Noradnata Kultura. Serria: Bio log i ia , Zemedenie i Lesovlistvo (Bul l , .chambre Cult . Nation. Ser: B i o l . A g r i c , et S i l v i c u l t . ) 1: 239-240. 1946. 50. Barnett, H .L . and V.G. L i l l y . Physiology of the Fungi. McGraw-Hill Book Co. Inc. 1950. 51. Mulder, E . G . Importance of copper and molybdenum i n the nutr i t ion of higher plants and micro-organisms Trace Elements i n Plant Physiology. Symposium. Chronica Botanica Co. Waltham, Mass., U.S.A. 1950. _ 82 _ it 52. Sakamura, T. Uber einige fttr die Kultur von Aspergillen notwindigen Schwermetalle und des Befreiungsvergahren de Nahrlosung von ibren Spuren. Jour. Fac. Sci. Hakkiado Imp. Univ. Sec. 4: 99-116. 1936. 5(3. Fries, N. 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Chromosome rejoining capacity with respect to breakage sensitivity to X-rays and thermal neutrons. Genetics 40: 460-475. 1955. 61. Duggar, B.M., ED. Biological Effects of Radiation, p.1119. McGraw-Hill Book Co. Inc., N.Y. 1936. 62. Steinberg, R.A. and C. Thorn. Chemical induction of genetic changes i n A s p e r g i l l i . Jour. Hered. 31: 61-63. 1940. 63. Stokes, J . L . et a l . Synthesis of pyridoxine by a , l pyridoxineless , , X-ray mutant of Neurospora s i t o t h i l a . Arch. Biochem. 2: 235-245. 1943. 


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