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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 HEMLOCK  1  by WALTER BRUCE GLENN DENYER  2  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n t h e Department of B i o l o g y and Botany  We accept t h i s t h e s i s as conforming t o the standard  r e q u i r e d from c a n d i d a t e s f o r t h e  degree o f MASTER OF ARTS  Members o f the Department o f Biology  and Botany  THE UNIVERSITY OF BRITISH COLUMBIA October, 1951  • C o n t r i b u t i o n No. from the D i v i s i o n o f F o r e s t B i o l o g y , S c i e n c e S e r v i c e , Department o f A g r i c u l t u r e , Ottawa, Canada. A t h e s i s submitted t o t h e Department o f B i o l o g y and Botany, U n i v e r s i t y o f 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 o f Master o f A r t s . Forest  Pathologist  Abstract  A canker on the main stem o f oppressed western hemlock (Tsuga h e t e r o n h y l l a (Rafn.) S a r g . ) , 1 t o 3 i n c h e s i n d i a meter a t b r e a s t h e i g h t , was found a t P o w e l l R i v e r and T u m o u r I s l a n d , B r i t i s h Columbia. ly  e l l i p t i c a l , w i t h a conspicuous r e s i n exudation i n the  e a r l y stages. of  The canker i s i r r e g u l a r -  The canker appears to be annual.  A  species  Cenhalosporium ( F u n g i Imperfect!, M o n i l i a l e s ) was  con-  s i s t e n t l y i s o l a t e d from cankers c o l l e c t e d a t P o w e l l R i v e r . The p a t h o g e n i c i t y o f the organism has not been proven t o date.  The growth and hyphal c h a r a c t e r s of the organism, and  the  temperature-growth  are  described.  investigated.  r e l a t i o n s o f the organism i n c u l t u r e  The c o n i d i a l apparatus o f the organism Attempts to produce the p e r f e c t stage i n  c u l t u r e were u n s u c c e s s f u l .  was  T a b l e o f Contents Page Introduction D e s c r i p t i o n o f the Canker  1  I s o l a t i o n o f the Organism  2  I n o c u l a t i o n Experiments  .  D e s c r i p t i o n o f the Organism....  3 5  Method o f Study  5  Growth C h a r a c t e r s  7  Hyphal C h a r a c t e r s  8  Temperature R e l a t i o n s  of the Organism  i n Culture  10  C o n i d i a l Apparatus  12  Attempts t o Produce the P e r f e c t  Stage  i n Culture  L4  Discussion  15  Acknowledgements References Plates  .  19 20 22 - 27  Introduction  Specimens o f a canker on oppressed western 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 l u m b i a , a t P o w e l l R i v e r , B« C. i n June, 1949.  C u l t u r e s o f a fungus were o b t a i n e d from 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  c i a t e d w i t h I t was b e g u n i n A p r i l ,  1950.  d i s t r i b u t i o n o f t h e organism, specimens  asso-  To d e t e r m i n e t h e  o f cankers were  c o l l e c t e d from western hemlock a t Campbell R i v e r , P o r t A l b e r n i a n d Cameron L a k e o n V a n c o u v e r  Island.  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 o r g a n i s m o b t a i n e d from Powell  River.  I n J u n e , 1 9 5 0 , 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 , w h e r e 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 oppressed western hemlock  was f o u n d 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  of a Douglas-^fir (Pseudotsuea t a x i f o l i a  consisted  (Poir) B r i t t . )  stand,  A5 t o 50 y e a r s o l d w i t h some w e s t e r n h e m l o c k , w e s t e r n r e d c e d a r (Thu.1 a p l i c a t a D. Don) a n d g r a n d f i r Lindley).  T h e 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 h e m l o c k w i t h  some r e d c e d a r a n d g r a n d f i r . locality.  (Abies grandis  Cankers were c o l l e c t e d i n t h i s  T h e o r g a n i s m i s o l a t e d f r o m t h e s e c a n k e r s was t h e  II  same as t h a t 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 b a s i s f o r the d e s c r i p t i o n o f the canker and i t s a s s o c i a t e d Further  organism.  c o l l e c t i o n s o f a canker on western hemlock  were made by Mr. E. G. Marples, F o r e s t e r , P o w e l l R i v e r Company, on T u m o u r I s l a n d , B. C. i n September,  1950.  When r e c e i v e d most o f t h e s e specimens were too d r y f o r s u c c e s s f u l c u l t u r i n g , but from two the o r i g i n a l organism was  obtained.  pure nemlock.  The f o r e s t cover on T u m o u r I s l a n d was  almost  The cankers came from oppressed m a t e r i a l  under s c a t t e r e d old-growth western hemlock and western r e d cedar.  1  D e s c r i p t i o n o f the Canker  Cankers o c c u r r e d on the main stem o f oppressed  western  hemlock, 1 t o 3 i n c h e s i n diameter a t b r e a s t h e i g h t . to L o r more cankers were found on each stem.  The  One  cankers  were i r r e g u l a r l y e l l i p t i c a l ( P I . I , F i g . 1 and 2 ) , 2 t o 6 inches i n length.  On  some t r e e s 2 o r more cankers had fused  to produce an e l o n g a t e , i r r e g u l a r canker that extended f o r 1 t o 2 f e e t along the stem. The f i r s t i n d i c a t i o n o f the presence of a canker was  a  conspicuous r e s i n e x u d a t i o n from the margins o f a s l i g h t l y depressed area o f bark.  The bark otherwise appeared  The bark a t the margin o f these cankers was but sometimes the canker was  often  normal.  unbroken,  o u t l i n e d by c r a c k i n g o f the  bark. On  some t r e e s a p p a r e n t l y a b o r t i v e cankers were found.  These appeared as a roughening o f the bark over a s m a l l c i r c u l a r a r e a up to 1 i n c h i n diameter, with a s m a l l amount of r e s i n exudation from the canker. I n l a t e r stages, the bark over the cankered a r e a  was  n o t i c e a b l y depressed and f i r m l y a t t a c h e d to the u n d e r l y i n g wood except' a t the margins where, due t o c a l l u s growth, bark was  the  cracked, r a i s e d , and i n the process o f b e i n g  sloughed o f f . There was no r e s i n exudation a t t h i s s t a g e . The i n n e r bark o f the cankers was  dark brown.  The margin o f  2  h e a l t h y and d i s e a s e d bark was marked by a dark narrow l i n e . The wood o f 1 or 2 annual r i n g s under the canker, i n some cases was gray, but i n most o f t h e cankers was n o t appre-^ c i a b l y d i f f e r e n t from normal wood. A l l cankers t h a t y i e l d e d t h e organism were a t l e a s t 2 y e a r s o l d , and most were o l d e r , s i n c e there were from 2 to 9 r i n g s o f c a l l u s growth a t the margin.  I t would  appear  t h a t the organism i s a c t i v e l y p a r a s i t i c f o r 1 y e a r o n l y . There was no evidence o f diebaek o f the c a l l u s , and a l l cankers examined had a smooth, curved margin o f c a l l u s growth. Cankers u s u a l l y had a branch stub a t the c e n t r e .  This  appeared t o be the p o i n t o f entrance o f the canker organism.  I s o l a t i o n o f the Organism  Two o f the 6 o r i g i n a l cankers c o l l e c t e d a t P o w e l l R i v e r , B. C. by D r . D. C. Buckland were c u l t u r e d .  From 9  p l a n t i n g s on malt agar, 6 produced pure c u l t u r e s o f Cephalosnorium sp. ( F u n g i Imperfect!, M o n i l i a l e s ) .  The  remainder were s t e r i l e . P l a n t i n g s on malt agar were made from the i n n e r bark near the margin o f 12 cankers from P o w e l l R i v e r . 7 p l a n t i n g s were made from each canker.  Three t o  Of t h e 52 c u l t u r e s  3  obtained:  26 were pure Cephalosporium^  12 were Cephalo-  sporium mixed w i t h some o t h e r organism, and the remainder comprised v a r i o u s molds and b a c t e r i a .  Cephalosporium was  i s o l a t e d from 10 o f t h e 12 cankers c u l t u r e d .  Cephalosporium  was t h e o n l y organism o b t a i n e d c o n s i s t e n t l y . I n s p i t e o f t h e i r d r i e d c o n d i t i o n , 2 cankers from T u m o u r I s l a n d were c u l t u r e d . were made from each canker.  Four p l a n t i n g s on m a l t agar T h i s r e s u l t e d i n 1 pure  culture  o f Cephalosporium from 1 canker, and 1 c u l t u r e o f Cephalosporium mixed w i t h some o t h e r organism from the o t h e r .  The  r e s t o f the p l a n t i n g s developed i n t o a v a r i e t y of molds.  I n o c u l a t i o n Experiments  Cephalosporium i s o l a t e d from the cankers was used t o i n o c u l a t e western hemlock t r e e s , 1 t o L inches i n diameter a t b r e a s t h e i g h t , a t the U n i v e r s i t y o f B r i t i s h Columbia i n May, 1950. Inoculum was prepared as f o l l o w s :  S t e r i l e i n n e r bark  c h i p s o f western hemlock were p l a c e d on malt agar i n P e t r i dishes.  The p l a t e s were i n o c u l a t e d w i t h Cephalosporium and  the c u l t u r e s allowed t o develop u n t i l the fungus had become w e l l e s t a b l i s h e d on the c h i p s . as inoculum.  These bark c h i p s were used  C o n t r o l s c o n s i s t e d o f s t e r i l e bark c h i p s and  A  malt agar o n l y . S i x t r e e s were i n o c u l a t e d on the main stem i n t h e f o l l o w i n g manner:  The a r e a t o be i n o c u l a t e d was swabbed  with 70 p e r c e n t a l c o h o l . ( P I . I , F i g . 3)  s  An i n v e r t e d "U-shaped" i n c i s i o n  about 1 i n c h square, p e n e t r a t i n g t o t h e  cambium, was made w i t h a s t e r i l e k n i f e b l a d e .  The f l a p  thus  formed was r a i s e d s l i g h t l y , and a p i e c e o f inoculum p l a c e d under the bark.  A moistened  s t e r i l e gauze pad was then  p l a c e d over the i n o c u l a t i o n , and f a s t e n e d t o t h e stem. S e v e r a l l a y e r s o f waxed paper Were bound over the pad t o r e t a i n moisture. I n o c u l a t i o n s were made over the stubs o f s m a l l branches c u t o f f f l u s h w i t h the bark, and under bark cont a i n i n g ho branch s t u b s .  Seventeen i n o c u l a t i o n s and 6  c o n t r o l s were made. The i n o c u l a t i o n s were examined a f t e r 3 months. A l l i n o c u l a t i o n s showed some degree o f fungus a c t i v i t y . o r white mycelium  Yellow;  was p r e s e n t i n t h e c u t s i n the bark.  On  the s m a l l e r t r e e s , t h e r e were s m a l l sunken areas i n t h e bark around t h e i n o c u l a t i o n s ( P I . 1, F i g . 3 ) . There was no evidence o f c a l l u s growth i n the i n o c u l a t i o n s , w h i l e c a l l u s growth was e v i d e n t i n t h e c o n t r o l s . C u l t u r a l t e s t s were made on 12 o f the i n o c u l a t i o n s from 5 o f t h e 6 i n o c u l a t e d t r e e s i n October, 1950, approxi m a t e l y 5 months a f t e r i n o c u l a t i o n .  S e v e r a l p l a n t i n g s were  5  made from each i n o c u l a t i o n .  Only f o u r pure c u l t u r e s o f  Cephalogpofi 11m, from 3 separate i n o c u l a t i o n s , were o b t a i n e d . As the above experiment was i n c o n c l u s i v e , f u r t h e r work on i n o c u l a t i o n i s being c a r r i e d out.  D e s c r i p t i o n o f the Organism  Method o f Study The t e r m i n o l o g y used by P i n k e r t o n (11) i n d e s c r i b i n g . /the f r u i t i n g  s t r u c t u r e s of Cephalosporium i s used h e r e .  - The cephalosporium i s the spore b a l l o r c o n i d i a l head, the cephalophore i s the e n t i r e f r u c t i f i c a t i o n ,  including  spore b a l l and c o n i d i o p h o r e , and the p h i a l i d e i s the end c e l l of a c o n i d i o p h o r e . Unless otherwise s t a t e d the organism was grown on malt agar, prepared a c c o r d i n g t o t h e f o l l o w i n g formula: D i f c o malt e x t r a c t  12.5  gm.  D i f c o Bacto-agar  20.0  gm.  D i s t i l l e d water  1000.0 c.c.  Ten c u l t u r e s o f Gephalospor 1 ^ i -m on malt agar i n P e t r i d i s h e s were used as the b a s i s f o r the d e s c r i p t i o n o f t h e gross f e a t u r e s  and growth r a t e o f the organism i n c u l t u r e .  The c u l t u r e s were prepared by p l a c i n g a p i e c e o f inoculum about 5 mm.  square, taken from near the advancing zone o f  6  an a c t i v e l y - g r o w i n g c u l t u r e , on the malt agar a t t h e edge of the d i s h . temperature  The c u l t u r e s were grown i n the dark a t room (12°  t o 20° C.) f o r 6 weeks.  They were brought  out f o r examination a t weekly i n t e r v a l s , a t which times the r a d i a l growth, appearance noted.  Photographs  and c o l o r o f the c u l t u r e s  were  were taken a t the end o f 2, L and 6  weeks ( P I . I I , F i g . 1 t o 4-) •  (10)  Nobles  method was f o l l o w e d  M u n s e l l (9)  f o r t h e d e s c r i p t i o n o f the c u l t u r e s .  color  e q u i v a l e n t s are g i v e n . Hyphae and c o n i d i o p h o r e s were examined, and camera l u c i d a drawings made by p l a c i n g a cover s l i p d i r e c t l y over a p o r t i o n o f the c u l t u r e on a P e t r i d i s h .  Cephalosporia  were d i f f i c u l t t o examine s i n c e any movement o f t h e c o n i d i o phore was s u f f i c i e n t t o d i s l o d g e the c o n i d i a .  Cephalophores  were observed under low power o f the microscope by d i r e c t examination o f c u l t u r e s , 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 o f t h e s t r u c t u r e were obscured by drops o f m o i s t u r e , which u s u a l l y formed around For  condensed  the c o n i d i a l heads.  c l o s e r examination of c e p h a l o s p o r i a , agar s l i d e c u l t u r e s  were prepared by p l a c i n g a drop o f s t e r i l e , melted p o t a t o d e x t r o s e agar on a s t e r i l e s l i d e , and immediately ing with conidia.  When the agar had begun t o s e t , a s t e r i l e  cover s l i p was p l a c e d over the drop o f agar. were incubated i n a moist chamber. u s u a l l y formed  inoculat-  The  cultures  C e p h a l o s p o r i a were  i n 2 t o L days, and could be examined under  7  the h i g h dry o b j e c t i v e . c u l t u r e s appeared  The c e p h a l o s p o r i a formed  i n such  t y p i c a l except t h a t fewer c o n i d i a were  formed per head than on c u l t u r e s i n P e t r i d i s h e s . Measurements were taken w i t h a f i l a r micrometer  from  c o n i d i a mounted i n 7 percent potassium hydroxide, w i t h a s m a l l amount of p h l o x i n e added. u s i n g the o i l immersion  objective.  Germination o f c o n i d i a was of  C o n i d i a were measured  observed by p l a c i n g a  drop  spore suspension on a s m a l l square o f s t e r i l e c e l l o p h a n e  on p o t a t o d e x t r o s e agar i n a P e t r i d i s h . p l a c e w i t h i n 2L hours.  The  Germination took  s l i p s o f cellophane were then  mounted on s l i d e s , and e i t h e r examined d i r e c t l y or s t a i n e d w i t h g e n t i a n v i o l e t (12). Repeated  attempts to observe the f o r m a t i o n o f c o n i d i a  i n agar s l i d e c u l t u r e s under the microscope were unsuccessful.  Growth C h a r a c t e r s The growth o f t h e organism i s slow, v a r y i n g from 2.5 4.1  cm.  i n 6 weeks (average, 3.6  F i g . 3 and A).  cnu)  to  ( P I . V I , F i g . 1; P I . I I ,  The advancing zone i s s l i g h t l y bayed, h y a l i n e  and appressed, extending 1 to 5 nim. beyond the a e r i a l mycelium. The mat  i s white and f a r i n a c e o u s , w i t h h y a l i n e d r o p l e t s ;  becoming y e l l o w (Munsell c o l o r e q u i v a l e n t s :  10.0 YR  8.0/6.0  at 2 weeks; 10.0 YR 7.5/6.5, 10.0 YR 8.0/7.5 a t L weeks; and  8  10.0 YR 7.0/7.0. a t 6 weeks) and s u b f e l t y i n o l d e r p a r t s o f the c u l t u r e ; o c c a s i o n a l l y with s e c t o r s o f f e l t y , y e l l o w i s h t o h y a l i n e a e r i a l mycelium  ( P i . I I , F i g . A)  to the advancing zone.  There i s no e f f e c t  from the inoculum on the agar.  The white and f a r i n a c e o u s p a r t s o f the mat  i n the  c u l t u r e s were f a i n t l y zonate ( P I . I I , F i g . 2 to A). zoning i s a p p a r e n t l y due to exposure t o l i g h t ,  test  The  since  c u l t u r e s grown i n the dark, without exposure to l i g h t a t intervals,  do not have a zonate  appearance.  Hyphal C h a r a c t e r s The hyphae have simple septa throughout.  The  advancing  zone i s composed o f h y a l i n e hyphae, 2 t o A microns i n d i a meter ( P I . I l l , F i g . 7 ) .  The a e r i a l mycelium  c o n s i s t s of:  Narrow hyphae, 2 t o A microns i n diameter ( P I . I l l , F i g . 6 ) , s i m i l a r t o the hyphae i n the advancing zone; broad h y a l i n e hyphae, A to 8 microns i n diameter, c y l i n d r i c Figi  (PI. I l l ,  2) t o s p h e r o i d ( P I . I l l , F i g . 3 ) , with the c e l l s  e n l a r g e d a t one end ( P I . I l l , F i g . 1)> p r o s t r a t e , o r from the s u r f a c e o f the agar f o r a p o r t i o n o f t h e i r  often rising  length  near the t i p s ; and s m a l l e r broad hyphae w i t h amber-colored contents ( P I . I l l , F i g . 4 ) . some c e l l s may  O c c a s i o n a l l y i n a broad hypha,  be c o l o r e d and o t h e r s h y a l i n e ( P I . I l l , F i g . 5 ) .  The amber-colored hyphae produce the c o l o r i n the c u l t u r e s . The c o n i d i a a r e borne i n r o u g h l y s p h e r i c a l heads  9  (cephalosporia)  ( P I . IV, F i g . 1; P I . V, F i g . .12), which are  7 to 27 microns i n diameter (average 11 microns).  The  c o n i d i a are o v a l to obovate, o c c a s i o n a l l y c y l i n d r i c , and  o n e - c e l l e d , 2.4- to L.5  by 4-.1  to 6.7  microns (average  o f 50 spores from a c u l t u r e on malt agar, 3.2 Mucous i s produced by the conidiophore, the l a r g e r c e p h a l o s p o r i a .  and  on Sax's medium (8) produce a compact mat,  measuring 2.3  t o 3.0  The  conidiophores  out may  hyphae, which may length.  phores and  slightly,  by .22.2  L4  microns).  or b i f u r c a t e ( P I . IV, F i g . 3) In older parts  are u s u a l l y compound (PI.: IV,  be simple,  and a r i s e from broad  be p r o s t r a t e or a e r i a l f o r p a r t of  Simple conidiophores,  hyphae, may  kernels  w i t h amber drops o f  p h i a l i d e s taper  a r i s e from p r o s t r a t e hyphae.  o f c u l t u r e s , the conidiophores F i g . 2 and 4)>  is visible in  a r i s e from p r o s t r a t e or a e r i a l hyphae.  Near the advancing zone, simple conidiophores  microns).  microns i n diameter at the base by  to 28 microns i n l e n g t h (average 2.6 The  by 5.2  C u l t u r e s grown on b a r l e y  mucous c o n t a i n i n g many spores.  hyaline  their  a r i s i n g from narrow p r o s t r a t e  a l s o occur i n o l d e r p a r t s o f c u l t u r e s .  Conidio-  c o n i d i a are produced p r o f u s e l y over the s u r f a c e  c u l t u r e s , except i n the h y a l i n e , f e l t y areas, under the growth c h a r a c t e r s , where they are  r e f e r r e d to sparse.  C o n i d i a germinate w i t h i n 24 hours, when p l a c e d on a n u t r i e n t medium.  The  c o n i d i a s w e l l to twice or t h r e e times  t h e i r o r i g i n a l s i z e and  send out 1 or 2 germ tubes.  of  10  Temperature R e l a t i o n s o f the Organism i n C u l t u r e  The growth o f the organism i n c u l t u r e , i n r e l a t i o n to temperature, was  s t u d i e d to f i n d the minimum, maximum and  optimum temperature f o r growth.  The growth r a t e s g i v e n are  the average r a d i a l growth o f 5 c u l t u r e s on malt agar i n P e t r i dishes (PI. VI, F i g . 2 ) .  P l a t e s were i n o c u l a t e d i n the  c e n t r e with a p i e c e o f inoculum about 5 mm.  square, taken  from near the advancing zone o f an a c t i v e l y - g r o w i n g on malt agar.  The c u l t u r e s were examined,  and  culture  growth  measurements recorded at weekly i n t e r v a l s f o r 3 weeks. S i n c e t h e r e was no change i n the t r e n d of growth over the three-week p e r i o d , the values g i v e n represent r a d i a l  growth  at the end o f 3 weeks.  Temperature v a r i a t i o n o f the i n c u -  b a t o r s was w i t h i n 1 ° C ,  with the e x c e p t i o n o f the I 2 C . t e s t , a  which tended to have a s l i g h t l y g r e a t e r v a r i a t i o n .  A  l i m i t e d number o f constant temperature i n c u b a t o r s was  avail-  a b l e , so t h a t a complete temperature range could not be t e s t e d a t one time. I n i t i a l l y c u l t u r e s were i n c u b a t e d a t : 20°,  25°  and 3 5 ° C  14°,  16° and 1 8 ° C ,  and a f o u r t h a t :  0 , 4°, O :  9°,  A second s e r i e s was i n c u b a t e d a t : a t h i r d at:  .20°,  22°  12°,  12°, 12°-,  H ° , 16°, 18° and 2 0 ° C ,  and 25°C.  There was no growth a t 0°  or at 3 0 ° C ,  indicating  the minimum temperature f o r growth l i e s between 0°  and  that  11  4°C., and t h e 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 o f an optimum temperature i n t h i s series.  first  However, s i n c e the r a t e o f growth i n c r e a s e d up t o  20°C., and d e c l i n e d a t 2 5 ° C ;  i t was thought t h a t the optimum  temperature l a y between 12° and 20°C. s e t up t o t e s t t h i s p o s s i b i l i t y .  The second s e r i e s was  The growth r a t e a t  1A°C,  i n the second s e r i e s , seemed t o be much too h i g h , and w e l l above the t r e n d o f growth a t a d j a c e n t temperatures ( P I . V I , F i g . 2 ) . T h i s was checked by the t h i r d growth a t 1A°  s e r i e s , i n which t h e  conformed w i t h the g e n e r a l t r e n d . There was  s t i l l a p o s s i b i l i t y t h a t the optimum temperature l a y between 20° and 25°C.  T h i s was i n v e s t i g a t e d i n the f o u r t h s e r i e s ,  which was i n c u b a t e d f o r l l days.  I n t h i s s e r i e s , growth was  most r a p i d a t 20°C, i n d i c a t i n g t h a t the optimum d i d not l i e above 20°C.  The optimum temperature, t h e r e f o r e , appears t o  l i e between 18°\ and 20°C. The mat o f c u l t u r e s i n c u b a t e d a t the lower temperatures (below 12°C.) was t h i n and appressed.  These c u l t u r e s remained  white, w i t h ho p r o d u c t i o n o f a y e l l o w c o l o r over the t h r e e week p e r i o d . abundant mat.  A t temperatures above 20°, growth was slow, but  a e r i a l mycelium was produced, --giving a compact  felty  There was no appressed advancing zone as a t lower  temperatures, and a e r i a l mycelium was p r e s e n t t o the l i m i t of growth. of  growth.  The mat i n such c u l t u r e s was c o l o r e d t o the l i m i t  12  C o n i d i a l Apparatus  P i n k e r t o n (11) i n v e s t i g a t e d the c o n i d i a l apparatus o f s e v e r a l human, p a r a s i t i c s p e c i e s o f Cephalosporium and some r e l a t e d Hyphomycetes.  The c o n i d i a l apparatus was found t o be  s i m i l a r i n the species i n v e s t i g a t e d . the to  The c o n i d i a l apparatus o f  organism under study was i n v e s t i g a t e d i n a s i m i l a r manner compare I t w i t h the s p e c i e s i n v e s t i g a t e d by P i n k e r t o n . Agar s l a n t s o f D i f c o Bacto potato dextrose agar i n  c a p s u l e v i a l s were i n o c u l a t e d w i t h C e p h a l o s p p - H inrij and i n cubated f o r 10 days a t 20°C. f i x e d w i t h Hermann's f l u i d  The c u l t u r e s were k i l l e d and  (5) a p p l i e d d i r e c t l y t o the s l a n t s .  The v i a l s were evacuated f o r 15 minutes i n a vacuum d e s s i c a t o r , immediately f o l l o w i n g t h e a p p l i c a t i o n o f the f i x i n g fluid.  The c u l t u r e s were k i l l e d  and f i x e d f o r 12 hours,  then washed i n s l o w l y running water f o r 2A hours.  The s l a n t s  were then c u t up i n t o b l o c k s , and dehydrated i n an e t h y l alcohol series.  The b l o c k s were then r u n through an a l c o h o l -  x y l e n e s e r i e s , and embedded i n F i s h e r TIssuemat  (m.p. 52°' t o  54°C.). S e c t i o n s were cut a t 3, 5, and 15 microns.  The s e c t i o n s  were s t a i n e d with Heidenhain's Iron-alum-haematoxylin a c c o r d i n g t o the f o l l o w i n g schedule:  (A)  H y d r a t i o n , ;20 p e r c e n t  Merck S u p e r o x o l 1 hour, running water 30 minutes, A p e r c e n t Iron alum 2 hours, 2 changes o f d i s t i l l e d water i n 4 minutes,  13  1/2 p e r c e n t q u i c k r i p e n e d 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 s e c t i o n s were then dehydrated i n  e t h y l a l c o h o l , c l e a r e d i n x y l e n e , and mounted i n Ganada "balsam. S e c t i o n s cut at 5 microns were most s a t i s f a c t o r y .  The  s e c t i o n s cut at 15 microns were too t h i c k f o r e i t h e r good s t a i n i n g o r s a t i s f a c t o r y o b s e r v a t i o n under the o i l immersion o b j e c t i v e , which was n e c e s s a r y t o see d e t a i l s o f s t r u c t u r e . C o n i d i a l f o r m a t i o n i s f i r s t i n d i c a t e d by a narrowing of  the cytoplasm, near the t i p o f the p h i a l i d e i n t o a s l e n d e r  thread, a t the end o f which i s formed a small b a l l o f c y t o plasm ( P I . V, F i g . 1 ) . a conidium (P.l.  T h i s s m a l l b a l l e n l a r g e s to become  V, F i g . 1 to 4 ) .  Cytoplasm i s a p p a r e n t l y  drawn through the t h r e a d i n t o the developing spore, causing it  to enlarge.  The end w a l l o f the p h i a l i d e appears t o be  t h i c k e r than the l a t e r a l w a l l s , and to c o n t a i n a pore, p e n e t r a t e d by the t h r e a d of cytoplasm.  After  considerable  enlargement, w h i l e s t i l l w i t h i n the p h i a l i d e , the spore i s extruded through the end w a l l .  The p r o t o p l a s m i c t h r e a d ,  connecting spore and p h i a l i d e , i s broken, and a w a l l i s formed around the conidium ( P I . V, F i g *  5).  Another spore  begins t o form, w h i l e the f i r s t - f o r m e d spore i s s t i l l attached to the t i p o f the p h i a l i d e ( P I . V, F i g . 6 ) .  The  u e x t r u s i o n o f the next spore pushes the f i r s t - f o r m e d aside.  The process continues forming a  The passage (PI.  one  cephalosporium.  o f a nucleus i n t o the d e v e l o p i n g spore  V, F i g . 7 ) , does not appear to take p l a c e when the  spore i s o f a p a r t i c u l a r s i z e , spore may  s i n c e i n some cases, the  be s m a l l , and have a n u c l e u s ; w h i l e i n o t h e r s , may  appear to be n e a r l y f u l l s i z e and not y e t possess one.  The  p h i a l i d e i s u n i n u c l e a t e . N u c l e a r d i v i s i o n ( P I . V, F i g . 8) and m i g r a t i o n o f a daughter n u c l e u s appears t o take p l a c e as the spores a r e produced, observed i n any  s i n c e more than 2 n u c l e i were not  phialide.  The cytoplasm o f the p h i a l i d e i s a t f i r s t without v a c u o l e s i of  dense,  Vacuoles appear, forming f i r s t  and  a t the base  the p h i a l i d e , as spores are produced and the cytoplasm  used up, u n t i l the p h i a l i d e c o n t a i n s s e v e r a l v a c u o l e s ( P I . V, Fig.  9).  Attempts  to Produce  the P e r f e c t Stage i n C u l t u r e  On 2 cankers r e c e i v e d from T u m o u r I s l a n d , B. small orange  apothecia occurred.  C,  An attempt was made t o  o b t a i n c u l t u r e s from the bark o f the cankers, and from apothecia.  None was  the  o b t a i n e d however, because of the d r i e d  c o n d i t i o n o f the specimens  when r e c e i v e d .  No  ascospores  15  were p r e s e n t i n the a p o t h e c i a .  S i n c e these were the o n l y  cankers oh which f r u i t i n g o c c u r r e d , i t was not p o s s i b l e t o t e l l whether the a p o t h e c i a were the p e r f e c t stage o f t h i s s p e c i e s o f Cephalosporium, o r the p e r f e c t stage o f a secondary organism. I n an attempt t o induce the p e r f e c t stage i n c u l t u r e , the organism was  grown on a v a r i e t y o f media, which  included:  oat, prune, potato-dextrose-peptone, and cornmeal agar; p o t a t o dextrose agar w i t h 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  u s i n g the i n n e r bark o f western hemlock, i n s t e a d o f was  tested.  sawdust  Sax's n u t r i e n t agar (8) and b a r l e y k e r n e l s  a l s o used as a medium. any o f the above media.  (l),  was  The p e r f e c t stage was n o t induced by The organism grew w e l l on a l l the  media except L e o n i a n ' s . C u l t u r e s i s o l a t e d from s e v e r a l cankers were p a i r e d . p a i r e d c u l t u r e s i n t e r m i n g l e d f r e e l y , but the p e r f e c t stage was n o t  formed.  Discussion  C h r i s t e n s e n (3) s t u d i e d a canker on balsam f i r balsamea (L.) M i l l . ) Cephalosporium.  (Abies  i n Minnesota, caused by a s p e c i e s o f  The d e s c r i p t i o n o f the canker i s v e r y  The  16  s i m i l a r to the one under c o n s i d e r a t i o n h e r e .  The author  wrote to Dr. C. M. C h r i s t e n s e n , P r o f e s s o r of P l a n t Pathol o g y , U n i v e r s i t y o f Minnesota, f o r check c u l t u r e s , but none were i n e x i s t e n c e .  There are some d i f f e r e n c e s between the  two organisms i n c u l t u r e .  I n d e s c r i b i n g the organism found  i n Minnesota, C h r i s t e n s e n r e p o r t s :  "On malt agar a t tem-  p e r a t u r e s from 20° to 30°C. i t produces a f a i n t l y zonate c u l t u r e of white, f l u f f y , a e r i a l mycelium,  ...."  "Prelimi-  n a r y temperature s t u d i e s i n d i c a t e t h a t i t w i l l not grow a t 0° and 3 5 ° C , 27° t o 30°C."  grows slowly a t 15°C., and most r a p i d l y a t  I n c u l t u r e s o f the organism found i n B r i t i s h  Columbia, a y e l l o w c o l o r i s a n o t i c e a b l e f e a t u r e a f t e r 1 week, e s p e c i a l l y a t temperatures from 20° to 30°C. no growth a t 0?C«, ^  u  There i s  t the maximum temperature f o r growth  l i e s between 25° and 30°C.  Growth i s q u i t e r a p i d a t 1 5 ° C ,  and the optimum l i e s between 18° and 20°C. I n d e s c r i b i n g the hyphal c h a r a c t e r s o f the Minnesota organism, C h r i s t e n s e n s t a t e s :  "The c o n i d i o p h o r e s taper to  a s l e n d e r t i p , on which 1 to 3 c o n i d i a are borne s i m u l t a n eously, e i t h e r on short s t e r i g m a t a or d i r e c t l y on the conidiophores.  Each sterigma c o n t i n u e s to produce  conidia,  and these remain c l u s t e r e d i n a r o u g h l y s p h e r i c a l head about the t i p o f the c o n i d i o p h o r e . " I n the organism found i n B r i t i s h Columbia, the c o n i d i a are produced  endogenously,  and a r e extruded, one a t a time through the t i p o f the  17  conidiophore.  No sterigmata a r e p r e s e n t .  ments o f the two organisms are:  C o n i d i a l measure-  2.8 to 5.7 microns i n  l e n g t h (average A.3 microns) f o r t h e Minnesota organism, compared w i t h A.l  t o 6.7 microns i n l e n g t h (average  microns) f o r the B r i t i s h Columbia organism.  5.2  I n view of the  above d i f f e r e n c e s , t h e two organisms cannot be c o n s i d e r e d identical. I n comparing c o n i d i a l f o r m a t i o n i n the s p e c i e s o f Cephalosporium i n v e s t i g a t e d by P i n k e r t o n (11), and t h i s organism, t h e r e a r e a g a i n some d i f f e r e n c e s .  However,  P i n k e r t o n made an e x t e n s i v e i n v e s t i g a t i o n o f s e v e r a l s p e c i e s u s i n g both t h e c e l l o i d i n and the p a r a f f i n method o f embedding c u l t u r e s , w h i l e o n l y the p a r a f f i n method was employed i n t h i s study.  I n t h i s connection, P i n k e r t o n r e p o r t s t h a t  cytoplasmic material.  accessory  i n c l u s i o n s were more n o t i c e a b l e i n t h e c e l l o i d i n T h i s may account f o r t h e f a c t t h a t c e r t a i n s t r u c -  t u r e s I n the p h i a l i d e were not observed material studied.  i n the p a r a f f i n  A c c o r d i n g , t o P i n k e r t o n , a c h a i n o f 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 i n t o s e v e r a l s m a l l rods a t the t i p o f the p h i a l i d e . form a r i g i d c o l l a r ,  These rods  so t h a t the end w a l l i s weaker than  the l a t e r a l w a l l s a t the t i p o f the p h i a l i d e .  As t h e spore  Is f o r c e d out, t h e middle o f the t i p i s s t r e t c h e d and broken. These granules and rods were not observed material studied.  i n the p a r a f f i n  P i n k e r t o n observes, however:  "Very t h i n  18  s e c t i o n s of p a r a f f i n m a t e r i a l seem to i n d i c a t e t h a t the spore i s a b s t r i c t e d as i s the y e a s t bud. may  be e x p l a i n e d by assuming t h a t one  T h i s appearance  of the g r a n u l e s  at  the t i p r e a l l y s t a y s i n the c e n t e r of the p h i a l i d e and draws the cytoplasm  as a narrow stream i n t o the spore;  i n some t h i n s e c t i o n s i t i s i m p o s s i b l e to see any granules."  T h i s appears to agree with the  made on spore f o r m a t i o n i n t h i s organism.  and  lateral  observations Pinkerton  found  t h a t the contents o f the p h i a l i d e cleave i n t o segments, which e v e n t u a l l y become spores.  The  cleavage may  be  only  p a r t i a l with the c l e a v e d p a r t s o c c u r r i n g o n l y i n s m a l l numbers towards the t i p of the p h i a l i d e .  No  cleavage of the contents of the p h i a l i d e was this  organism.  s u g g e s t i o n of observed  in  19  A cknowledg ement s  The  author wishes to express h i s a p p r e c i a t i o n t o Dr.  D. C. Buckland, 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 Columbia f o r d i r e c t i o n i n t h i s to Dr. C. G. R i l e y , O f f i c e r - i n - C h a r g e , Dominion  study;  Laboratory  o f F o r e s t Pathology, Saskatoon; t o P r o f e s s o r T. C. V a n t e r p o o l , P r o f e s s o r o f P l a n t Pathology, U n i v e r s i t y o f S a s k a t c h ewan; t o Dr. R. H. H a s k i n s , P r a i r i e R e g i o n a l L a b o r a t o r y o f the N a t i o n a l Research C o u n c i l , f o r t e c h n i c a l a d v i c e ; t o Mr. E . G. Marples, F o r e s t e r , P o w e l l R i v e r Company, f o r the c o l l e c t i o n o f canker specimens and i n f o r m a t i o n on f o r e s t types;  t o Mr. B r i a n Leddy o f the P r a i r i e R e g i o n a l  Laboratory  of t h e N a t i o n a l Research C o u n c i l ; and Mr. J . E . P a t t e r s o n , Dominion L a b o r a t o r y  o f F o r e s t Pathology, Saskatoon, f o r  photographic a s s i s t a n c e .  20  References  1.  Badcock, E . C.  New methods f o r the c u l t i v a t i o n o f  wood-rotting f u n g i . 25: 200-205. 2.  Trans. B r i t . Mycol. Soc.  1941-  B a r n e t t , H. L., and V. G. L i l l y .  The e f f e c t o f  b i o t i n upon the formation and development o f p e r i t h e c i a , a s c i and ascospores by S o r d a r i a f i m i c o l a Ces. and de Not. 3.  C h r i s t e n s e n , C. M. fir.  4.  Phytopath.  Am. J . B o t .  Cephalosporium 27: 788-791.  procedures  used by the b i o l o g i c a l s t a i n commission,  p. I I B - 8 .  Lee, B o l l e s .  1946.  The m i c r o t o m i s t s vade-mecum, 1  Lee, B o l l e s .  1  L e o n i a n , L. H.  8.  A study o f f a c t o r s promoting  pycnidium  Am. J . B o t . 11:  1924.  M i l l e r , E . C.  Plant physiology,  Book Company I n c . , New York. 9.  p. 152.  1950.  f o r m a t i o n i n some s p h a e r o p s i d a l e s . 19-50.  p. 32.  1950.  The m i c r o t o m i s t s vade-mecum,  The B l a k i s t o n Co., P h i l a d e l p h i a . 7.  canker o f balsam  Staining  The B l a k i s t o n Co., P h i l a d e l p h i a . 6.  1947.  1937.  Conn, H. J . , and M. A. Darrow.  B i o t e c k P u b l i c a t i o n s , Geneva. 5.  34: 196-204.  M u n s e l l book o f c o l o r . Baltimore.  1929.  p. 195.  McGraw-Hill  1931.  M u n s e l l C o l o r Company I n c .  21  10.  N o b l e s , M. K.  Studies i n forest pathology.  I d e n t i f i c a t i o n of cultures  of wood-rotting  C a n . J . R e s . C 26: 2 8 1 - 4 3 1 . 11.  P i n k e r t o n , M. E .  mycetes. 12.  Yuill,  aspergilli.  study of c o n i d i a l  a n d some r e l a t e d  A n n . Mo. B o t . Gdn.  E.  fungi.  194-8.  A comparative  formation i n Cephalosporium  VI  23 ( l ) : 1-68.  The n u m b e r s o f n u c l e i i n c o n i d i a B r i t . Mycol. Soc.  33: 3 2 4 - 3 3 1 .  hypho1936. of 1950.  22  Plate I  The F i g . 1.  Canker  A n a t u r a l canker on the main stem o f western hemlock (Tsuga h e t e r o p h y l l a X  F i g . 2.  1/2.  The  same canker shown i n F i g . 1 w i t h the bark  removed, showing the bark. F i g . 3.  (Rafn.) Sarg.) .  X  the extent o f the canker under  1/2.  A canker on the main stem o f western hemlock, 5 months a f t e r i n o c u l a t i o n .  X  1/2.  23  Plate II  Cephalosporium  sp. on M a l t Agar, Grown i n the Dark i n 90 mm.  Fig.  P e t r i Dishes  1.  Growth a f t e r 2 weeks.  F i g . 2.  Growth a f t e r L weeks,  Fig.  3.  Growth a f t e r 6 weeks.  Fig.  k.  Growth a f t e r 6 weeks, with a h y a l i n e on t h e l e f t .  sector  P  L  A  T  E  31  24  Plate III  Camera L u c l d a Drawings  o f the Organism  from C u l t u r e s  on  M a l t Agar  Fig.  1.  Broad h y a l i n e hyphae, c e l l s w i t h swollen ends,  Fig.  2.  Broad h y a l i n e hypha w i t h r e g u l a r  Fig.  3.  Broad h y a l i n e hypha w i t h i r r e g u l a r  Fig.  L.  Narrow hypha w i t h amber-colored c o n t e n t s .  Fig.  5.  Hypha w i t h a narrow, amber-colored c e l l ,  cells. cells.  and a broad h y a l i n e c e l l w i t h bases o f conidiophores. Fig.  6.  Narrow h y a l i n e hypha.  Fig.  7.  Hyphae from the advancing zone.  PLATE  III  25  P l a t e IV  Camera L u c i d a Drawings o f t h e Organism  F i g . 1.  Cephalophores  from an agar s l i d e  culture  on p o t a t o d e x t r o s e agar. F i g . 2.  Compound c o n i d i o p h o r e , w i t h c o n i d i a l heads removed, from a c u l t u r e on malt agar.  Fig.  3.  C o n i d i o p h o r e s , w i t h c o n i d i a l heads removed, from near t h e advancing zone o f a c u l t u r e on malt agar.  F i g . A.  Compound c o n i d i o p h o r e , w i t h c o n i d i a l heads removed, from a c u l t u r e on malt agar.  P L A T E EZT  3  4-  26  Plate V  Camera L u c i d a Drawings o f C o n i d i a l F o r m a t i o n i n Cephalosporium  sp.  F i g . 1-3. Development o f a conidium w i t h i n the t i p o f the phialide. F i g . A.  E x t r u s i o n o f the spore through the t i p o f the  Fig.  5.  phialide.  Phialide,  showing  the c o n n e c t i o n between  conidium and p h i a l i d e F i g . 6.  broken.  Mature conidium, w i t h another spore b e g i n n i n g t o form i n the t i p o f the p h i a l i d e .  F i g . 7.  Passage o f a nucleus i n t o 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 v a c u o l e s .  F i g . 10.  P h i a l i d e w i t h spore broken o f f , and a new  spore  b e g i n n i n g t o form. F i g . 11.  Conidia.  Fig.  Photomicrograph  12.  on malt agar.  o f cephalophores from a X  200.  culture  PLATE  V  PLATE GROWTH OF CEPHALOSPORIUM SP. ON MALT AGAR AT ROOM TEMPERATURE AVERAGE OF »  CULTURES  FIG. I  VI  

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