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Cephalosporium Sp., an organism associated with a canker of western hemlock Denyer, Walter Bruce Glenn 1951

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CEPHALOSPORIUM SP. ' AN ORGANISM ASSOCIATED WITH A CANKER OF WESTERN HEMLOCK1 by WALTER BRUCE GLENN DENYER2 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of Biology and Botany We accept t h i s thesis as conforming to the standard required from candidates f o r the degree of MASTER OF ARTS Members of the Department of Biology and Botany THE UNIVERSITY OF BRITISH COLUMBIA October, 1951 •Contribution No. from the D i v i s i o n of Forest Biology, Science Service, Department of Agriculture, Ottawa, Canada. A thesis submitted to the Department of Biology and Botany, University of B r i t i s h Columbia i n p a r t i a l f u l f i l m e n t f o r the degree of Master of Arts. Forest Pathologist Abstract A canker on the main stem of oppressed western hemlock (Tsuga heteronhylla (Rafn.) Sarg.), 1 to 3 inches i n d i a -meter at breast height, was found at Powell River and Tumour Island, B r i t i s h Columbia. The canker i s i r r e g u l a r -l y e l l i p t i c a l , with a conspicuous r e s i n exudation i n the ea r l y stages. The canker appears to be annual. A species of Cenhalosporium (Fungi Imperfect!, Moniliales) was con-s i s t e n t l y i s o l a t e d from cankers col l e c t e d at Powell River. The pathogenicity of the organism has not been proven to date. The growth and hyphal characters of the organism, and the temperature-growth relations of the organism i n culture are described. The c o n i d i a l apparatus of the organism was investigated. Attempts to produce the perfect stage i n culture were unsuccessful. Table of Contents Page Introduction Description of the Canker 1 I s o l a t i o n of the Organism 2 Inoculation Experiments . 3 Description of the Organism.... 5 Method of Study 5 Growth Characters 7 Hyphal Characters 8 Temperature Relations of the Organism i n Culture 10 Coni d i a l Apparatus 12 Attempts to Produce the Perfect Stage i n Culture L4 Discussion 15 Acknowledgements . 19 References 20 Plates 22 - 27 I n t r o d u c t i o n Specimens o f a c a n k e r on o p p r e s s e d w e s t e r n hemlock (Tsuga h e t e r o p h y l l a (Rafn.) Sarg.) were c o l l e c t e d by D r . D. C. B u c k l a n d , A s s o c i a t e P r o f e s s o r o f F o r e s t P a t h o l o g y , U n i v e r s i t y o f B r i t i s h C o lumbia, a t P o w e l l R i v e r , B« C. i n Ju n e , 1949. C u l t u r e s o f a fungus were o b t a i n e d f r o m t h e c a n k e r s , b u t t h e o r g a n i s m was n o t i d e n t i f i e d . An I n v e s t i g a t i o n o f t h e c a n k e r and t h e o r g a n i s m a s s o -c i a t e d w i t h I t was begun i n A p r i l , 1950. To d e t e r m i n e t h e d i s t r i b u t i o n o f t h e or g a n i s m , specimens o f c a n k e r s were c o l l e c t e d f r o m w e s t e r n hemlock a t Ca m p b e l l R i v e r , P o r t A l b e r n i and Cameron L a k e on Vancouver I s l a n d . These cankers were c u l t u r e d , b u t none y i e l d e d t h e or g a n i s m o b t a i n e d from P o w e l l R i v e r . I n J u n e , 1950, a v i s i t was made t o t h e e x p e r i m e n t a l f o r e s t a t P o w e l l R i v e r , where t h e c a n k e r was f i r s t r e p o r t e d . Here a f a i r l y heavy i n f e c t i o n o f o p p r e s s e d w e s t e r n hemlock was found i n a s m a l l l o c a l i z e d a r e a . The o v e r s t o r y c o n s i s t e d o f a D o u g l a s - ^ f i r ( P s e u d o t s u e a t a x i f o l i a ( P o i r ) B r i t t . ) s t a n d , A5 t o 50 y e a r s o l d w i t h some w e s t e r n hemlock, w e s t e r n r e d ce d a r (Thu.1 a p l i c a t a D. Don) and grand f i r ( A b i e s g r a n d i s L i n d l e y ) . The u n d e r s t o r y was c h i e f l y w e s t e r n hemlock w i t h some r e d c e d a r and grand f i r . Cankers were c o l l e c t e d i n t h i s l o c a l i t y . The o r g a n i s m i s o l a t e d f r om t h e s e c a n k e r s was t h e II same as that o r i g i n a l l y obtained by Dr. Buckland. This c o l l e c t i o n formed the basis f o r the des c r i p t i o n of the canker and i t s associated organism. Further c o l l e c t i o n s of a canker on western hemlock were made by Mr. E. G. Marples, Forester, Powell River Company, on Tumour Island, B. C. i n September, 1950. When received most of these specimens were too dry f o r successful culturing, but from two the o r i g i n a l organism was obtained. The f o r e s t cover on Tumour Island was almost pure nemlock. The cankers came from oppressed material under scattered old-growth western hemlock and western red cedar. 1 Description of the Canker Cankers occurred on the main stem of oppressed western hemlock, 1 to 3 inches i n diameter at breast height. One to L or more cankers were found on each stem. The cankers were i r r e g u l a r l y e l l i p t i c a l (PI. I, F i g . 1 and 2), 2 to 6 inches i n length. On some trees 2 or more cankers had fused to produce an elongate, i r r e g u l a r canker that extended f o r 1 to 2 feet along the stem. The f i r s t i n d i c a t i o n of the presence of a canker was a conspicuous r e s i n exudation from the margins of a s l i g h t l y depressed area of bark. The bark otherwise appeared normal. The bark at the margin of these cankers was often unbroken, but sometimes the canker was outlined by cracking of the bark. On some trees apparently abortive cankers were found. These appeared as a roughening of the bark over a small c i r c u l a r area up to 1 inch i n diameter, with a small amount of r e s i n exudation from the canker. In l a t e r stages, the bark over the cankered area was noticeably depressed and firm l y attached to the underlying wood except' at the margins where, due to cal l u s growth, the bark was cracked, raised, and i n the process of being sloughed o f f . There was no r e s i n exudation at t h i s stage. The inner bark of the cankers was dark brown. The margin of 2 healthy and diseased bark was marked by a dark narrow l i n e . The wood of 1 or 2 annual rings under the canker, i n some cases was gray, but i n most of the cankers was not appre-^ c i a b l y d i f f e r e n t from normal wood. A l l cankers that yielded the organism were at l e a s t 2 years old, and most were older, since there were from 2 to 9 rings of ca l l u s growth at the margin. I t would appear that the organism i s a c t i v e l y p a r a s i t i c for 1 year only. There was no evidence of diebaek of the c a l l u s , and a l l cankers examined had a smooth, curved margin of c a l l u s growth. Cankers usually had a branch stub at the centre. This appeared to be the point of entrance of the canker organism. I s o l a t i o n of the Organism Two of the 6 o r i g i n a l cankers collected at Powell River, B. C. by Dr. D. C. Buckland were cultured. From 9 plantings on malt agar, 6 produced pure cultures of Cephalosnorium sp. (Fungi Imperfect!, M o n i l i a l e s ) . The remainder were s t e r i l e . Plantings on malt agar were made from the inner bark near the margin of 12 cankers from Powell River. Three to 7 plantings were made from each canker. Of the 52 cultures 3 obtained: 26 were pure Cephalosporium^ 12 were Cephalo-sporium mixed with some other organism, and the remainder comprised various molds and bacteria. Cephalosporium was is o l a t e d from 10 of the 12 cankers cultured. Cephalosporium was the only organism obtained consistently. In s p i t e of t h e i r dried condition, 2 cankers from Tumour Island were cultured. Four plantings on malt agar were made from each canker. This resulted i n 1 pure culture of Cephalosporium from 1 canker, and 1 culture of Cephalo-sporium mixed with some other organism from the other. The rest of the plantings developed into a va r i e t y of molds. Inoculation Experiments Cephalosporium i s o l a t e d from the cankers was used to inoculate western hemlock trees, 1 to L inches i n diameter at breast height, at the University of B r i t i s h Columbia i n May, 1950. Inoculum was prepared as follows: S t e r i l e inner bark chips of western hemlock were placed on malt agar i n P e t r i dishes. The plates were inoculated with Cephalosporium and the cultures allowed to develop u n t i l the fungus had become well established on the chips. These bark chips were used as inoculum. Controls consisted of s t e r i l e bark chips and A malt agar only. Six trees were inoculated on the main stem i n the following manner: The area to be inoculated was swabbed with 70 percent alcohol. An inverted "U-shaped" i n c i s i o n (PI. I, F i g . 3)s about 1 inch square, penetrating to the cambium, was made with a s t e r i l e knife blade. The f l a p thus formed was raised s l i g h t l y , and a piece of inoculum placed under the bark. A moistened s t e r i l e gauze pad was then placed over the inoculation, and fastened to the stem. Several layers of waxed paper Were bound over the pad to ret a i n moisture. Inoculations were made over the stubs of small branches cut o f f f l u s h with the bark, and under bark con-taining ho branch stubs. Seventeen inoculations and 6 controls were made. The inoculations were examined a f t e r 3 months. A l l inoculations showed some degree of fungus a c t i v i t y . Yellow; or white mycelium was present i n the cuts i n the bark. On the smaller trees, there were small sunken areas i n the bark around the inoculations (PI. 1, F i g . 3). There was no evidence of call u s growth i n the inoculations, while c a l l u s growth was evident i n the controls. C u l t u r a l tests were made on 12 of the inoculations from 5 of the 6 inoculated trees i n October, 1950, approx-imately 5 months a f t e r inoculation. Several plantings were 5 made from each inoculation. Only four pure cultures of Cephalogpofi 11m, from 3 separate inoculations, were obtained. As the above experiment was inconclusive, further work on inoculation i s being ca r r i e d out. Description of the Organism Method of Study The terminology used by Pinkerton (11) i n describing . /the f r u i t i n g structures of Cephalosporium i s used here. - The cephalosporium i s the spore b a l l or c o n i d i a l head, the cephalophore i s the enti r e f r u c t i f i c a t i o n , including spore b a l l and conidiophore, and the phi a l i d e i s the end c e l l of a conidiophore. Unless otherwise stated the organism was grown on malt agar, prepared according to the following formula: Di f c o malt extract 12.5 gm. Difco Bacto-agar 20.0 gm. D i s t i l l e d water 1000.0 c.c. Ten cultures of Gephalospor 1 i^-m on malt agar i n P e t r i dishes were used as the basis f o r the description of the gross features and growth rate of the organism i n culture. The cultures were prepared by placing a piece of inoculum about 5 mm. square, taken from near the advancing zone of 6 an actively-growing culture, on the malt agar at the edge of the dish. The cultures were grown i n the dark at room temperature (12° to 20° C.) f o r 6 weeks. They were brought out f o r examination at weekly i n t e r v a l s , at which times the r a d i a l growth, appearance and color of the cultures were noted. Photographs were taken at the end of 2, L and 6 weeks (PI. I I , F i g . 1 to 4-) • Nobles (10) method was followed f o r the description of the cultures. Munsell (9) color equivalents are given. Hyphae and conidiophores were examined, and camera l u c i d a drawings made by placing a cover s l i p d i r e c t l y over a portion of the culture on a P e t r i dish. Cephalosporia were d i f f i c u l t to examine since any movement of the conidio-phore was s u f f i c i e n t to dislodge the conidia. Cephalophores were observed under low power of the microscope by d i r e c t examination of cultures, but t h i s was not s a t i s f a c t o r y as d e t a i l s of the structure were obscured by drops of condensed moisture, which usually formed around the c o n i d i a l heads. For closer examination of cephalosporia, agar s l i d e cultures were prepared by placing a drop of s t e r i l e , melted potato dextrose agar on a s t e r i l e s l i d e , and immediately inoculat-ing with conidia. When the agar had begun to set, a s t e r i l e cover s l i p was placed over the drop of agar. The cultures were incubated i n a moist chamber. Cephalosporia were usu a l l y formed i n 2 to L days, and could be examined under 7 the high dry objective. The cephalosporia formed i n such cultures appeared t y p i c a l except that fewer conidia were formed per head than on cultures i n P e t r i dishes. Measurements were taken with a f i l a r micrometer from conidia mounted i n 7 percent potassium hydroxide, with a small amount of phloxine added. Conidia were measured using the o i l immersion objective. Germination of conidia was observed by placing a drop of spore suspension on a small square of s t e r i l e cellophane on potato dextrose agar i n a P e t r i dish. Germination took place within 2L hours. The s l i p s of cellophane were then mounted on s l i d e s , and either examined d i r e c t l y or stained with gentian v i o l e t (12). Repeated attempts to observe the formation of conidia i n agar s l i d e cultures under the microscope were unsuccess-f u l . Growth Characters The growth of the organism i s slow, varying from 2.5 to 4.1 cm. i n 6 weeks (average, 3.6 cnu) (PI. VI, F i g . 1; PI. I I , F i g . 3 and A). The advancing zone i s s l i g h t l y bayed, hyaline and appressed, extending 1 to 5 nim. beyond the a e r i a l mycelium. The mat i s white and farinaceous, with hyaline droplets; becoming yellow (Munsell color equivalents: 10.0 YR 8.0/6.0 at 2 weeks; 10.0 YR 7.5/6.5, 10.0 YR 8.0/7.5 at L weeks; and 8 10.0 YR 7.0/7.0. at 6 weeks) and subfelty i n older parts of the culture; occasionally with sectors of f e l t y , yellowish to hyaline a e r i a l mycelium ( P i . I I , F i g . A) from the inoculum to the advancing zone. There i s no ef f e c t on the agar. The white and farinaceous parts of the mat i n the test cultures were f a i n t l y zonate (PI. I I , F i g . 2 to A). The zoning i s apparently due to exposure to l i g h t , since cultures grown i n the dark, without exposure to l i g h t at i n t e r v a l s , do not have a zonate appearance. Hyphal Characters The hyphae have simple septa throughout. The advancing zone i s composed of hyaline hyphae, 2 to A microns i n d i a -meter (PI. I l l , F i g . 7). The a e r i a l mycelium consists of: Narrow hyphae, 2 to A microns i n diameter (PI. I l l , F i g . 6), s i m i l a r to the hyphae i n the advancing zone; broad hyaline hyphae, A to 8 microns i n diameter, c y l i n d r i c (PI. I l l , F i g i 2) to spheroid (PI. I l l , F i g . 3), with the c e l l s often enlarged at one end (PI. I l l , F i g . 1)> prostrate, or r i s i n g from the surface of the agar f o r a portion of t h e i r length near the t i p s ; and smaller broad hyphae with amber-colored contents (PI. I l l , F i g . 4). Occasionally i n a broad hypha, some c e l l s may be colored and others hyaline (PI. I l l , F i g . 5). The amber-colored hyphae produce the color i n the cultures. The conidia are borne i n roughly spherical heads 9 (cephalosporia) (PI. IV, F i g . 1; PI. V, F i g . .12), which are 7 to 27 microns i n diameter (average 11 microns). The conidia are oval to obovate, occasionally c y l i n d r i c , hyaline and one-celled, 2.4- to L.5 by 4-.1 to 6.7 microns (average of 50 spores from a culture on malt agar, 3.2 by 5.2 microns). Mucous i s produced by the conidiophore, and i s v i s i b l e i n the larger cephalosporia. Cultures grown on barley kernels on Sax's medium (8) produce a compact mat, with amber drops of mucous containing many spores. The phialides taper s l i g h t l y , measuring 2.3 to 3.0 microns i n diameter at the base by L4 to 28 microns i n length (average 2.6 by .22.2 microns). The conidiophores a r i s e from prostrate or a e r i a l hyphae. Near the advancing zone, simple or bifurcate (PI. IV, F i g . 3) conidiophores a r i s e from prostrate hyphae. In older parts of cultures, the conidiophores are usually compound (PI.: IV, F i g . 2 and 4)> out may be simple, and a r i s e from broad hyphae, which may be prostrate or a e r i a l f o r part of t h e i r length. Simple conidiophores, a r i s i n g from narrow prostrate hyphae, may also occur i n older parts of cultures. Conidio-phores and conidia are produced profusely over the surface of cultures, except i n the hyaline, f e l t y areas, referred to under the growth characters, where they are sparse. Conidia germinate within 24 hours, when placed on a nutrient medium. The conidia swell to twice or three times t h e i r o r i g i n a l size and send out 1 or 2 germ tubes. 10 Temperature Relations of the Organism i n Culture The growth of the organism i n culture, i n r e l a t i o n to temperature, was studied to f i n d the minimum, maximum and optimum temperature f o r growth. The growth rates given are the average r a d i a l growth of 5 cultures on malt agar i n P e t r i dishes (PI. VI, F i g . 2 ) . Plates were inoculated i n the centre with a piece of inoculum about 5 mm. square, taken from near the advancing zone of an actively-growing culture on malt agar. The cultures were examined, and growth measurements recorded at weekly i n t e r v a l s f o r 3 weeks. Since there was no change i n the trend of growth over the three-week period, the values given represent r a d i a l growth at the end of 3 weeks. Temperature v a r i a t i o n of the incu-bators was within 1 ° C , with the exception of the I 2 aC. test, which tended to have a s l i g h t l y greater v a r i a t i o n . A l i m i t e d number of constant temperature incubators was a v a i l -able, so that a complete temperature range could not be tested at one time. I n i t i a l l y cultures were incubated at: 0 O : , 4 ° , 9 ° , 1 2 ° , 20° , 25° and 3 5°C A second series was incubated at: 12°-, 1 4 ° , 16° and 1 8 ° C , a t h i r d at: 1 2 ° , H ° , 16°, 18° and 2 0 °C, and a fourth at: .20°, 22° and 25°C. There was no growth at 0° or at 3 0 ° C , i n d i c a t i n g that the minimum temperature f o r growth l i e s between 0° and 11 4°C., and the maximum between 25° and 30°C. There was no d e f i n i t e i n d i c a t i o n of an optimum temperature i n t h i s f i r s t s e r i e s . However, since the rate of growth increased up to 20°C., and declined at 25°C; i t was thought that the optimum temperature lay between 12° and 20°C. The second series was set up to t e s t t h i s p o s s i b i l i t y . The growth rate at 1A°C, i n the second series, seemed to be much too high, and well above the trend of growth at adjacent temperatures (PI. VI, F i g . 2). This was checked by the t h i r d series, i n which the growth a t 1A° conformed with the general trend . There was s t i l l a p o s s i b i l i t y that the optimum temperature l a y between 20° and 25°C. This was investigated i n the fourth s e r i e s , which was incubated f o r l l days. In t h i s series, growth was most rapid at 20°C, i n d i c a t i n g that the optimum did not l i e above 20°C. The optimum temperature, therefore, appears to l i e between 18°\ and 20°C. The mat of cultures incubated at the lower temperatures (below 12°C.) was t h i n and appressed. These cultures remained white, with ho production of a yellow color over the three-week period. At temperatures above 20°, growth was slow, but abundant a e r i a l mycelium was produced, --giving a compact f e l t y mat. There was no appressed advancing zone as at lower temperatures, and a e r i a l mycelium was present to the l i m i t of growth. The mat i n such cultures was colored to the l i m i t of growth. 12 Conidial Apparatus Pinkerton (11) investigated the co n i d i a l apparatus of several human, p a r a s i t i c species of Cephalosporium and some rel a t e d Hyphomycetes. The c o n i d i a l apparatus was found to be s i m i l a r i n the species investigated. The c o n i d i a l apparatus of the organism under study was investigated i n a s i m i l a r manner to compare I t with the species investigated by Pinkerton. Agar slants of Difco Bacto potato dextrose agar i n capsule v i a l s were inoculated with Cephalospp-H inrij and i n -cubated f o r 10 days at 20°C. The cultures were k i l l e d and f i x e d with Hermann's f l u i d (5) applied d i r e c t l y to the slants. The v i a l s were evacuated for 15 minutes i n a vacuum dessi -cator, immediately following the application of the f i x i n g f l u i d . The cultures were k i l l e d and fixed f o r 12 hours, then washed i n slowly running water f o r 2A hours. The slants were then cut up into blocks, and dehydrated i n an ethyl alcohol series. The blocks were then run through an alcohol-xylene series, and embedded i n Fisher TIssuemat (m.p. 52°' to 54°C.). Sections were cut at 3, 5, and 15 microns. The sections were stained with Heidenhain's Iron-alum-haematoxylin (A) according to the following schedule: Hydration, ;20 percent Merck Superoxol 1 hour, running water 30 minutes, A percent Iron alum 2 hours, 2 changes of d i s t i l l e d water i n 4 minutes, 13 1/2 percent quick ripened haematoxylin (6) 12 hours, 3 changes of tap water i n 2 minutes, 2 percent i r o n alum 5 to 10 minutes, 2 changes of d i s t i l l e d water i n 10 minutes, and running water 1 hour. The sections were then dehydrated i n ethyl alcohol, cleared i n xylene, and mounted i n Ganada "balsam. Sections cut at 5 microns were most s a t i s f a c t o r y . The sections cut at 15 microns were too thick f o r either good staining or s a t i s f a c t o r y observation under the o i l immersion objective, which was necessary to see d e t a i l s of structure. C o n i d i a l formation i s f i r s t indicated by a narrowing of the cytoplasm, near the t i p of the p h i a l i d e into a slender thread, at the end of which i s formed a small b a l l of cyto-plasm (PI. V, F i g . 1). This small b a l l enlarges to become a conidium (P.l. V, F i g . 1 to 4). Cytoplasm i s apparently drawn through the thread into the developing spore, causing i t to enlarge. The end wall of the phialide appears to be thicker than the l a t e r a l walls, and to contain a pore, penetrated by the thread of cytoplasm. After considerable enlargement, while s t i l l within the ph i a l i d e , the spore i s extruded through the end wall. The protoplasmic thread, connecting spore and p h i a l i d e , i s broken, and a wall i s formed around the conidium (PI. V, F i g * 5). Another spore begins to form, while the first-formed spore i s s t i l l attached to the t i p of the phialide (PI. V, F i g . 6). The u extrusion of the next spore pushes the first-formed one aside. The process continues forming a cephalosporium. The passage of a nucleus into the developing spore (PI. V, F i g . 7), does not appear to take place when the spore i s of a p a r t i c u l a r size, since i n some cases, the spore may be small, and have a nucleus; while i n others, may appear to be nearly f u l l size and not yet possess one. The p h i a l i d e i s uninucleate. Nuclear d i v i s i o n (PI. V, F i g . 8) and migration of a daughter nucleus appears to take place as the spores are produced, since more than 2 n u c l e i were not observed i n any p h i a l i d e . The cytoplasm of the ph i a l i d e i s at f i r s t dense, and without vacuoles i Vacuoles appear, forming f i r s t at the base of the ph i a l i d e , as spores are produced and the cytoplasm used up, u n t i l the phialide contains several vacuoles (PI. V, F i g . 9 ) . Attempts to Produce the Perfect Stage i n Culture On 2 cankers received from Tumour Island, B. C , small orange apothecia occurred. An attempt was made to obtain cultures from the bark of the cankers, and from the apothecia. None was obtained however, because of the dried condition of the specimens when received. No ascospores 15 were present i n the apothecia. Since these were the only cankers oh which f r u i t i n g occurred, i t was not possible to t e l l whether the apothecia were the perfect stage of t h i s species of Cephalosporium, or the perfect stage of a secondary organism. In an attempt to induce the perfect stage i n culture, the organism was grown on a v a r i e t y of media, which included: oat, prune, potato-dextrose-peptone, and cornmeal agar; potato dextrose agar with b i o t i n (2), Leonian's medium (7), and Badcock's medium ( l ) . Badcock f r u c t i f i c a t i o n f l a s k s ( l ) , using the inner bark of western hemlock, instead of sawdust was tested. Sax's nutrient agar (8) and barley kernels was also used as a medium. The perfect stage was not induced by any of the above media. The organism grew well on a l l the media except Leonian's. Cultures i s o l a t e d from several cankers were paired. The paired cultures intermingled f r e e l y , but the perfect stage was not formed. Discussion Christensen (3) studied a canker on balsam f i r (Abies  balsamea (L.) M i l l . ) i n Minnesota, caused by a species of Cephalosporium. The description of the canker i s very 16 s i m i l a r to the one under consideration here. The author wrote to Dr. C. M. Christensen, Professor of Plant Patho-logy, University of Minnesota, f o r check cultures, but none were i n existence. There are some differences between the two organisms i n culture. In describing the organism found i n Minnesota, Christensen reports: "On malt agar at tem-peratures from 20° to 30°C. i t produces a f a i n t l y zonate culture of white, f l u f f y , a e r i a l mycelium, ...." " P r e l i m i -nary temperature studies indicate that i t w i l l not grow at 0° and 3 5 ° C , grows slowly at 15°C., and most rapidl y at 27° to 30°C." In cultures of the organism found i n B r i t i s h Columbia, a yellow color i s a noticeable feature a f t e r 1 week, es p e c i a l l y at temperatures from 20° to 30°C. There i s no growth at 0?C«, ^ u t the maximum temperature f o r growth l i e s between 25° and 30°C. Growth i s quite rapid at 15°C, and the optimum l i e s between 18° and 20°C. In describing the hyphal characters of the Minnesota organism, Christensen states: "The conidiophores taper to a slender t i p , on which 1 to 3 conidia are borne simultan-eously, either on short sterigmata or d i r e c t l y on the conidiophores. Each sterigma continues to produce conidia, and these remain clustered i n a roughly spherical head about the t i p of the conidiophore." In the organism found i n B r i t i s h Columbia, the conidia are produced endogenously, and are extruded, one at a time through the t i p of the 17 conidiophore. No sterigmata are present. C o n i d i a l measure-ments of the two organisms are: 2.8 to 5.7 microns i n length (average A.3 microns) f o r the Minnesota organism, compared with A.l to 6.7 microns i n length (average 5.2 microns) f o r the B r i t i s h Columbia organism. In view of the above differences, the two organisms cannot be considered i d e n t i c a l . In comparing c o n i d i a l formation i n the species of Cephalosporium investigated by Pinkerton (11), and t h i s organism, there are again some differences. However, Pinkerton made an extensive i n v e s t i g a t i o n of several species using both the c e l l o i d i n and the p a r a f f i n method of embedding cultures, while only the p a r a f f i n method was employed i n th i s study. In t h i s connection, Pinkerton reports that accessory cytoplasmic inclusions were more noticeable i n the c e l l o i d i n material. This may account f o r the f a c t that c e r t a i n struc-tures In the phi a l i d e were not observed i n the p a r a f f i n material studied. According,to Pinkerton, a chain of small round granules c o l l e c t l o n g i t u d i n a l l y , and merge into several small rods at the t i p of the phiali d e . These rods form a r i g i d c o l l a r , so that the end wall i s weaker than the l a t e r a l walls at the t i p of the phialide. As the spore Is forced out, the middle of the t i p i s stretched and broken. These granules and rods were not observed i n the p a r a f f i n material studied. Pinkerton observes, however: "Very t h i n 18 sections of p a r a f f i n material seem to indicate that the spore i s abstricted as i s the yeast bud. This appearance may be explained by assuming that one of the granules at the t i p r e a l l y stays i n the center of the p h i a l i d e and draws the cytoplasm as a narrow stream into the spore; and i n some thi n sections i t i s impossible to see any l a t e r a l granules." This appears to agree with the observations made on spore formation i n t h i s organism. Pinkerton found that the contents of the phialide cleave into segments, which eventually become spores. The cleavage may be only p a r t i a l with the cleaved parts occurring only i n small numbers towards the t i p of the p h i a l i d e . No suggestion of cleavage of the contents of the phialide was observed i n t h i s organism. 19 A cknowledg ement s The author wishes to express h i s appreciation to Dr. D. C. Buckland, Associate Professor of Forest Pathology, U n i v e r s i t y of B r i t i s h Columbia f o r d i r e c t i o n i n t h i s study; to Dr. C. G. Riley, Officer-in-Charge, Dominion Laboratory of Forest Pathology, Saskatoon; to Professor T. C. Vanter-pool, Professor of Plant Pathology, University of Saskatch-ewan; to Dr. R. H. Haskins, P r a i r i e Regional Laboratory of the National Research Council, f o r technical advice; to Mr. E. G. Marples, Forester, Powell River Company, f o r the c o l l e c t i o n of canker specimens and information on f o r e s t types; to Mr. Brian Leddy of the P r a i r i e Regional Laboratory of the National Research Council; and Mr. J . E. Patterson, Dominion Laboratory of Forest Pathology, Saskatoon, f o r photographic assistance. 20 References 1. Badcock, E. C. New methods f o r the c u l t i v a t i o n of wood-rotting fungi. Trans. B r i t . Mycol. Soc. 25: 200-205. 1941-2. Barnett, H. L., and V. G. L i l l y . The e f f e c t of b i o t i n upon the formation and development of peritheci a , a s c i and ascospores by Sordaria f i m i c o l a Ces. and de Not. Am. J . Bot. 34: 196-204. 1947. 3. Christensen, C. M. Cephalosporium canker of balsam f i r . Phytopath. 27: 788-791. 1937. 4. Conn, H. J . , and M. A. Darrow. Staining procedures used by the b i o l o g i c a l s t a i n commission, p. IIB-8. Bioteck Publications, Geneva. 1946. 5. Lee, B o l l e s . The microtomist 1s vade-mecum, p. 32. The Blakiston Co., Philadelphia. 1950. 6. Lee, B o l l e s . The microtomist 1 s vade-mecum, p. 152. The Blakiston Co., Philadelphia. 1950. 7. Leonian, L. H. A study of factors promoting pycnidium formation i n some sphaeropsidales. Am. J . Bot. 11: 19-50. 1924. 8. M i l l e r , E. C. Plant physiology, p. 195. McGraw-Hill Book Company Inc., New York. 1931. 9. Munsell book of color. Munsell Color Company Inc. Baltimore. 1929. 21 10. N o b l e s , M. K. S t u d i e s i n f o r e s t p a t h o l o g y . V I I d e n t i f i c a t i o n o f c u l t u r e s o f w o o d - r o t t i n g f u n g i . Can. J . Res. C 26: 281-431. 194-8. 11. P i n k e r t o n , M. E. A c o m p a r a t i v e s t u d y o f c o n i d i a l f o r m a t i o n i n C e p h a l o s p o r i u m and some r e l a t e d hypho-mycetes. Ann. Mo. B o t . Gdn. 23 ( l ) : 1-68. 1936. 12. Y u i l l , E. The numbers o f n u c l e i i n c o n i d i a o f a s p e r g i l l i . B r i t . M y c o l . Soc. 33: 324-331. 1950. 22 Pl a t e I The Canker F i g . 1. A natural canker on the main stem of western hemlock (Tsuga heterophylla (Rafn.) Sarg.) . X 1/2. F i g . 2. The same canker shown i n F i g . 1 with the bark removed, showing the extent of the canker under the bark. X 1/2. F i g . 3. A canker on the main stem of western hemlock, 5 months a f t e r inoculation. X 1/2. 2 3 P l a t e II Cephalosporium sp. on Malt Agar, Grown i n the Dark i n 90 mm. P e t r i Dishes F i g . 1. F i g . 2. F i g . 3. F i g . k. Growth Growth Growth Growth on the af t e r 2 a f t e r L a f t e r 6 a f t e r 6 l e f t . weeks. weeks, weeks. weeks, with a hyaline sector P L A T E 31 24 P l a t e I I I Camera Luclda Drawings of the Organism from Cultures on Malt Agar F i g . 1. Broad hyaline hyphae, c e l l s with swollen ends, F i g . 2. Broad hyaline hypha with regular c e l l s . F i g . 3. Broad hyaline hypha with i r r e g u l a r c e l l s . F i g . L . Narrow hypha with amber-colored contents. F i g . 5. Hypha with a narrow, amber-colored c e l l , and a broad hyaline c e l l with bases of conidiophores. F i g . 6 . Narrow hyaline hypha. F i g . 7 . Hyphae from the advancing zone. P L A T E III 25 Plate IV Camera Lucida Drawings of the Organism F i g . 1. Cephalophores from an agar s l i d e culture on potato dextrose agar. F i g . 2. Compound conidiophore, with c o n i d i a l heads removed, from a culture on malt agar. F i g . 3. Conidiophores, with c o n i d i a l heads removed, from near the advancing zone of a culture on malt agar. F i g . A. Compound conidiophore, with c o n i d i a l heads removed, from a culture on malt agar. PLATE EZT 3 4-26 Plate V Camera Lucida Drawings of Conidial Formation i n Cephalosporium sp. F i g . 1-3. Development of a conidium within the t i p of the p h i a l i d e . F i g . A. Extrusion of the spore through the t i p of the p h i a l i d e . F i g . 5. P h i a l i d e , showing the connection between conidium and phialide broken. F i g . 6 . Mature conidium, with another spore beginning to form i n the t i p of the phialide. F i g . 7. Passage of a nucleus into a conidium. F i g . 8. Nuclear d i v i s i o n i n a p h i a l i d e . F i g . 9. P h i a l i d e , showing vacuoles. F i g . 10. P h i a l i d e with spore broken o f f , and a new spore beginning to form. F i g . 11. Conidia. F i g . 12. Photomicrograph of cephalophores from a culture on malt agar. X 200. P L A T E V P L A T E VI GROWTH OF CEPHALOSPORIUM SP. ON MALT AGAR AT ROOM TEMPERATURE AVERAGE OF » CULTURES F I G . I 

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