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Some seed-borne fungi of Dougals fir and their effect on germinating seed Cockerill, John 1959

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SOME SEED-BORNE FUNGI OF DOUGLAS FIR AND THEIR EFFECT ON GERMINATING SEED.  by  JOHN COCKERILL  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF FORESTRY i n t h e Department of Forestry  We a c c e p t t h i s t h e s i s a s conforming t o t h e s t a n d a r d 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 FORESTRY  Members o f t h e Department o f  THE UNIVERSITY OF BRITISH COLUMBIA  April  1959  - i i-  ABSTRACT  Seven seed l o t s o f Douglas f i r seed which became h e a v i l y contamina t e d w i t h mold d u r i n g g e r m i n a t i o n t e s t s a l s o had low g e r m i n a t i o n v a l u e s . A number o f f u n g i were i s o l a t e d from t h e seed c o a t and from w i t h i n s u r f a c e - s t e r i l i z e d seed sampled from t h e s e seed  lots.  S e v e r a l o f t h e f u n g i were found t o be capable o f a t t a c k i n g t h e r a d i c l e s o f g e r m i n a t i n g seeds under t h e c o n d i t i o n s o f t h e p a t h o g e n i c i t y tests.  Some o f t h e f u n g i , b o t h p a t h o g e n i c and non-pathogenic,  inhibited  the g e r m i n a t i o n o f seed t r e a t e d w i t h t h e spores o r mycelium o f t h e f u n g i during a period o f s t r a t i f i c a t i o n . The r e s u l t s o b t a i n e d i n d i c a t e t h a t t h e lower g e r m i n a t i o n v a l u e s o f t h e h e a v i l y contaminated  seed l o t s were due, i n p a r t a t l e a s t , t o t h e  a c t i v i t y o f t h e f u n g i capable o f a t t a c k i n g t h e g e r m i n a t i n g seed and t o f u n g i which i n h i b i t e d t h e s p r o u t i n g o f t h e seed by o t h e r means. Other f a c t o r s r e l a t i n g t o t h e t r e a t m e n t  o f t h e seed p r i o r t o t h e  g e r m i n a t i o n t e s t s which would a d v e r s e l y a f f e c t t h e g e r m i n a t i o n a r e d i s cussed i n r e l a t i o n t o t h i s  study.  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the requirements f o r an advanced degree at the  University  o f B r i t i s h Columbia, I agree t h a t the  L i b r a r y s h a l l make  it  study.  freely  a v a i l a b l e f o r reference  and  I further  agree t h a t permission f o r extensive copying of t h i s f o r s c h o l a r l y purposes may  be granted by the Head of  Department or by h i s r e p r e s e n t a t i v e s .  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  gain  s h a l l not be allowed without my  n r P i R  t.ry  The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada. Date  pflt.h April, 1959.  Columbia,  my  I t i s understood  that  FaculfaW$MM&ffl&&Xo£ F  thesis  written  financial  permission.  Index  Page Acknowledgements . *  •  i  Abstract  i i  Introduction  1  M a t e r i a l s and Methods  4  1.  I s o l a t i n g the f u n g i  4  2.  Identification  5  3.  Pathogenicity tests  5  4.  I n o c u l a t i o n o f s t r a t i f i e d seed  7  Experimental Results  7  1.  Identifications  7  2.  I n c i d e n c e and Occurrence o f Seed-borne F u n g i  13  3.  Pathogenicity tests  16  4.  Inoculation of s t r a t i f i e d  seed  17  Discussion  19  Summary and C o n c l u s i o n s  23  Bibliography  24  Tables 1.  Number and percentage o f i s o l a t e s o b t a i n e d e x t e r n a l l y  from  s i x seed l o t s 2.  3.  4.  5.  Number and percentage o f i s o l a t e s o b t a i n e d i n t e r n a l l y seven seed l o t s  13 from 14  P e r c e n t a g e o f seed i n each seed l o t from which a t l e a s t one i s o l a t e was o b t a i n e d  14  O c c u r r e n c e o f f u n g i i n t h e d i f f e r e n t seed l o t s . E x t e r n a l and i n t e r n a l i s o l a t i o n s a r e i n d i c a t e d  . . .  15  O c c u r r e n c e o f t h e p a t h o g e n i c f u n g i i n t h e seven seed l o t s  . . .  16  Page 6.  R e s u l t s o f p a t h o g e n i c i t y t e s t s a t the end o f a f i v e day period  7.  R e s u l t s o f g e r m i n a t i o n t e s t s w i t h spore i n o c u l a t e d seeds  8.  Summary o f the p a t h o g e n i c i t y and i n h i b i t i n g e f f e c t o f t h e isolated fungi  16 . . .  Plate I F i g u r e 1.  18  19 28  Method o f p l a n t i n g the seed t o i s o l a t e the f u n g i . Douglas f i r s e e d on m a l t agar i n a P e t r i  dish.  F i g u r e 2.  Growth o f f u n g i from seed p l a c e d on malt a g a r .  E i g u r e 3«  S u r f a c e s t e r i l i z e d seed g e r m i n a t i n g on m a l t a g a r .  F i g u r e s 4 and 5. Methods o f t e s t i n g t h e p a t h o g e n i c i t y o f the f u n g i . F i g . 4. S u r f a c e s t e r i l i z e d germinates p l a c e d a t v a r y i n g d i s t a n c e s from the edge o f a m y c e l i a l mat o f a fungus. F i g . 5« S u r f a c e s t e r i l i z e d germinates on a m y c e l i a l mat. F i g u r e 6.  Germinates whose r a d i c l e s have been a t t a c k e d by a p a t h o g e n i c fungus.  F i g u r e 7.  Germinates on the m y c e l i a l mat o f a non-pathogenic fungus. The r a d i c l e s have n o t been a t t a c k e d by the fungus.  ACKNOWLEDGEMENTS  The w r i t e r g r a t e f u l l y acknowledges t h e guidance and encouragement g i v e n by t h e l a t e Dr. D. C. B u c k l a n d , who suggested t h e problem and under whose d i r e c t i o n t h i s s t u d y was u n d e r t a k e n . He wishes t o thank Dean G. S. A l l e n f o r h i s i n t e r e s t and a d v i c e i n c o n n e c t i o n w i t h t h e problem and h i s h e l p f u l c r i t i c i s m and a d v i c e i n regard to the manuscript. To Dr. F . D i c k s o n who v e r y k i n d l y undertook  to review the t h e s i s  f o l l o w i n g Dr. Buckland's death and t o D r . J . E . B i e r , t h e a u t h o r to express h i s s i n c e r e  wishes  thanks.  T h i s study was made p o s s i b l e t h r o u g h a l e a v e o f absence  arranged  by Dr. J . E . B i e r , f o r m e r l y A s s o c i a t e C h i e f , F o r e s t B i o l o g y D i v i s i o n , Canada Department o f A g r i c u l t u r e , and f i n a n c i a l a s s i s t a n c e p r o v i d e d b y Dr. B u c k l a n d .  SOME SEED BORNE FUNGI OF DOUGLAS FIR THEIR EFFECT ON GERMINATING SEED.  AND  INTRODUCTION  The  g e r m i n a t i o n o f seed can be a f f e c t e d by the c o n d i t i o n s under which  seed i s s t o r e d , and the c o n d i t i o n o f the seed w h i l e i t i s i n s t o r a g e . and E l i a s o n  (15)  s t u d i e d the i n j u r y caused t o r e d p i n e d u r i n g the c l e a n i n g  p r o c e s s and found t h a t seed which had been i n j u r e d o r weakened by o r f l u c t u a t i n g temperatures Tervet  w i t h A s p e r g i l l u s spp. was growth o f the fungus.  contaminated  reduced by s t o r a g e c o n d i t i o n s which f a v o u r e d  Twenty-seven p e r c e n t o f the seed w i t h a  p e r cent was  the seed w i t h a m o i s t u r e  wetting  had a lower v i g o u r than u n i n j u r e d seed.  (29), found t h a t the g e r m i n a t i o n r a t e o f soybean seeds  c o n t e n t o f 6.6  Heit  the  moisture  f r e e o f i n f e c t i o n whereas o n l y 6 p e r c e n t o f  c o n t e n t o f 17.6  per cent was  F r o s t - i n j u r e d seed showed a h i g h e r percentage m i n a t i o n than u n i n j u r e d seed.  I t was  f r e e of i n f e c t i o n .  o f i n f e c t i o n and a lower  a l s o found t h a t a severe  ger-  retardation  i n s e e d l i n g growth r e s u l t e d from s t o r a g e under c o n d i t i o n s f a v o u r i n g the development o f A s p e r g i l l u s  spp.  The p o s s i b i l i t y o f a t t a c k by s e e d - b o r n e . f u n g i  i s a l s o i n c r e a s e d by  improper  h a n d l i n g o f the seed d u r i n g e x t r a c t i o n and  s t o r a g e and a l s o by  improper  d i s i n f e s t a t i o n methods which cause i n j u r y to the seed c o a t .  Hurd (16) found t h a t s a p r o p h y t i c f u n g i , p a r t i c u l a r l y P e n i c i l l i u m spp.  and  Rhizopus n i g r i c a n s , c o u l d r e a d i l y a t t a c k wheat and b a r l e y seed when the seed coat had been broken whereas seeds w i t h undamaged seed c o a t s were not a f f e c t e d by t h e s e f u n g i . copper  When the seed was  i n j u r e d by d i s i n f e s t i n g with,  s u l p h a t e o r formaldehyde f o l l o w e d by d r y i n g i t was  r e a d i l y attacked  by t h e s e same f u n g i , r e g a r d l e s s o f the c o n d i t i o n o f the seed c o a t .  -.2 Most s e e d - c o n t a m i n a t i n g f u n g i may be c o n t r o l l e d by d i r e c t sterilization seed ( l ) .  (10,  21),  surface  o r b y p r o v i d i n g a s c r e e n o f f u n g i c i d e above t h e  However some f u n g i a r e a b l e t o g a i n e n t r a n c e  i n t o t h e seed a t  v a r i o u s stages o f i t s development and, as t h e s e f u n g i w i l l n o t be removed by  s u r f a c e s t e r i l i z a t i o n , they may d e v e l o p under f a v o u r a b l e  a t t a c k the g e r m i n a t i n g  seed.  Koehler  c o n d i t i o n s and  (18) found t h a t Fusarium  S h e l d . emend. Snyder and Hansen c o u l d p e n e t r a t e  developing  moniliforme  e a r s o f c o r n and  Andrus (5)  contaminate t h e k e r n e l s b y growing through t h e s i l k s .  showed  t h a t Macrophomina'phaseoli (Maubl.) Ashby, t h e c a u s a l agent o f stem b l i g h t o f bean, was c a r r i e d w i t h i n the seed and would a t t a c k t h e s e e d l i n g p l a n t as i t developed.  Several fungi, including species of A l t e r n a r i a .  Stemphylium.  Fusarium. F e n i c i l l i u m . and Chaetomium were found t o be c a r r i e d i n t h e seed o f r e d and subterranean  c l o v e r by C h i l t o n ( 9 ) ,  and a s p e c i e s o f Rhizopus  was i s o l a t e d from w i t h i n s u r f a c e - s t e r i l i z e d seed by T o o l e and Drummond  (31).  F u n g i which a t t a c k young s e e d l i n g s i n the e a r l y stages o f growth, when t h e t i s s u e s a r e s u c c u l e n t , a r e c o l l e c t i v e l y termed d a m p i n g - o f f 1  1  fungi.  These f u n g i have been s t u d i e d i n c o n s i d e r a b l e d e t a i l because o f the l o s s e s they  cause i n c o n i f e r o u s seed beds I n f o r e s t n u r s e r i e s .  and T i n t  (30) were concerned w i t h  Roth and R i k e r (28)  c o n d i t i o n s under which s p e c i e s o f Pythium.  R h i z o c t o n i a . Phythophora. B o t r y t i s , Phomopsis. C o r t i c i u m . Mucor and Fusarium would a t t a c k w e l l - d e v e l o p e d  seedlings.  H a r t l e y , e t a l . (14),  i n work on  damping-off c l a s s i f i e d the d i s e a s e under t h r e e h e a d i n g s :  (1)  post-emergence,  i . e . a t t a c k i n g s e e d l i n g s which had emerged from t h e s o i l ;  (2)  pre-emergence  l o s s , i . e . where the r a d i c l e s were k i l l e d b e f o r e t h e s e e d l i n g s emerged; and (3)  decay o f dormant seed, i . e . l o s s o f t h e seed through f u n g a l a c t i o n  before  germination.  They suggested t h a t i n the l a t t e r case some o f t h e  seed c o u l d have been d e s t r o y e d by organisms o t h e r t h a n those  causing  damp-  - 3 -  ing o f f . Rathbun-Gravatt  (26), by i n o c u l a t i o n , found t h a t s t r a i n s o f Pythium,  R h i z o c t o n i a , and Fusarium were a b l e t o a t t a c k t h e r a d i c l e s o f g e r m i n a t i n g seed and would a l s o d e c r e a s e t h e s p r o u t i n g o f the seed.  I n general, the  f u n g i which d e c r e a s e d t h e s p r o u t i n g were t h e ones t h a t were a b l e t o decay the r a d i c l e s . Fisher  (12)  s t u d i e d the e f f e c t on g e r m i n a t i o n and seed decay o f  organisms i s o l a t e d from f r e s h c o n i f e r o u s seeds, from seeds removed from n u r s e r y beds where g e r m i n a t i o n was a f a i l u r e and from s t r a t i f i e d  seeds.  S u r f a c e - s t e r i l i z e d seed from these t r e a t m e n t s was cut open and i s o l a t i o n s were made from p o r t i o n s i n s i d e t h e s e e d c o a t ; t h e s u r f a c e o f the endosperm; i n s i d e the. endosperm; and t h e embryo. A s p e r g i l l u s . Gladosporium. Fusarium spp., Mucor. P e n i c i l l i u m . Rhizopus. Trichoderma, and s e v e r a l u n i d e n t i f i e d f u n g i were i s o l a t e d f r e s h seeds o f P i n u s b a n k s i a n a . P. ponderosa and P. r e s i n o s a ;  from  Alternaria.  A s p e r g i l l u s . B o t r y t i s , Fusarium, Mucor. P e n i c i l l i u m . R h i z o c t o n i a . Rhizopus. Trichoderma, and V e r t i c i l l i u m from seed which had f a i l e d t o germinate i n t h e seed beds; and A s p e r g i l l u s . Chaetonium globosum. Fusarium m o n i l i f o r m e . P e n i c i l l i u m , Rhizopus. T h i e l a v i a . and Trichoderma from s t r a t i f i e d  seed.  Some o f t h e s e organisms were used i n i n o c u l a t i o n experiments i n which g e r m i n a t i n g seeds were exposed  to cultures of the various f u n g i .  The  r e s u l t s o f t h e experiments showed t h a t a number o f the organisms  could  attack- the emerging  r a d i c l e s , o t h e r s d i d n o t cause a c o n s i s t e n t l o s s , and  some f a i l e d t o a t t a c k the r a d i c l e s .  I t was n o t e d t h a t , even when t h e r e  was no v i s i b l e r a d i c l e o r seed decay, t h e r e was a g e n e r a l d e c r e a s e i n t h e percentage g e r m i n a t i o n o f t h e i n o c u l a t e d  seed.  I n a s e r i e s o f g e r m i n a t i o n t e s t s c a r r i e d o u t by Dean G. S. A l l e n a t the U n i v e r s i t y o f B r i t i s h Columbia,  i t was f o u n d t h a t some seed l o t s ,  which  -  4  -  became h e a v i l y contaminated w i t h mold d u r i n g the t e s t s , had lower g e r m i n a t i o n v a l u e s t h a n seed which was  f r e e o f mold.  T h i s s t u d y was undertaken t o  determine i f the lower g e r m i n a t i o n i n these seed l o t s c o u l d be to  attributed  the a c t i v i t y , e i t h e r d i r e c t o r i n d i r e c t , o f the f u n g i c a r r i e d by the seed.  M a t e r i a l s and Methods  1.  I s o l a t i n g the F u n g i One hundred and f i f t y  Douglas f i r seeds were t a k e n from each o f s i x  seed l o t s , Uos. 4803, 4901, 4902, 5008, 5009 and 5010, which had been h e a v i l y contaminated w i t h molds d u r i n g g e r m i n a t i o n tests'.  The  fungi  c a r r i e d on the seed were i s o l a t e d by p l a c i n g the seed on two p e r c e n t malt agar i n P e t r i d i s h e s ( F i g . 1 ) . the  Ten seeds were spaced around the edge o f  d i s h and p r e s s e d i n t o the agar to p r o v i d e a m o i s t environment  development  o f the f u n g i .  The P e t r i - d l s h e s were k e p t i n the dark a t room  temperature and examined d a i l y f o r a f i v e - d a y p e r i o d . developed on the agar ( F i g . 2) In  f o r the  Ihen a  mycelium  a t r a n s f e r was made t o a malt a g a r  slant.  o r d e r t o avoid, a m i x t u r e o f s e v e r a l f u n g i from one seed o n l y the  fungus t o develop was  transferred.  first  The i s o l a t e s from each seed l o t were  retained f o r identification. To i s o l a t e the f u n g i c a r r i e d i n t e r n a l l y , a second sample o f seed taken from each seed l o t , seed, No. or  200  51GT.  seeds.  was  w i t h one a d d i t i o n a l sample from a seventh l o t o f  The number o f seeds i n each sample v a r i e d , b e i n g 50, The t o t a l sample c o n s i s t e d o f e l e v e n hundred  seeds.  150 The  seed samples were s u r f a c e - s t e r i l i z e d a c c o r d i n g to the method o f Muskett Malone (22).  Each seed sample was  and  p l a c e d i n a c o t t o n gauze bag, d i p p e d  b r i e f l y i n 95 p e r cent methyl a l c o h o l , t o reduce the s u r f a c e t e n s i o n ,  and  -  t h e n immersed i n a 1 : 1000  c o n t a i n i n g two The  per  -  s o l u t i o n of mercuric c h l o r i d e f o r f i v e minutes.  A f t e r s u r f a c e - s t e r i l i z i n g , the d i s t i l l e d water and  5  seed was  washed i n two  changes o f  sterile  t r a n s f e r r e d , under a s e p t i c c o n d i t i o n s , to P e t r i  dishes  cent m a l t a g a r .  P e t r i dishes  containing  the  s u r f a c e - s t e r i l i z e d seed were t r e a t e d  i n a s i m i l a r manner t o t h o s e w i t h the u n s t e r i l i z e d seed. Some seeds, a f t e r b e i n g embryos were removed and  s u r f a c e - s t e r i l i z e d were cut open and  plated.  i n t h i s manner were not r e c o r d e d  The  fungi developing  the  from seed t r e a t e d  s e p a r a t e l y from t h o s e d e v e l o p i n g  from  the  unopened seed. I t would be  e x p e c t e d t h a t the f u n g i i s o l a t e d from the seeds would show  some v a r i a t i o n depending on the method o f i s o l a t i o n u s e d . f u n g i i s o l a t e d i n t h i s study do not n e c e s s a r i l y r e p r e s e n t fungus f l o r a o f Douglas f i r seed, b u t i s o l a t e d by  2.  the method d e s c r i b e d  the  the  complete  o n l y the f u n g i which have been  above.  Identification The  f u n g i i s o l a t e d from the  o f the t e x t u r e and  seed were examined and  c o l o u r o f the m y c e l i a l mat,  m y c e l i a l mat  c h a r a c t e r i s t i c s f o l l o w s t h a t o f Nobles  Pathogenicity In order  grouped on the  basis  growth r a t e , r e a c t i o n on malt  agar, type o f spores and h y p h a l c h a r a c t e r i s t i c s .  3.  Therefore  The  d e s c r i p t i o n of  the  (24).  Tests  to determine which o f the f u n g i were c a p a b l e o f  g e r m i n a t i n g seed, i t was  n e c e s s a r y t o c a r r y out p a t h o g e n i c i t y  each o f . t h e f u n g i i s o l a t e d from the g e r m i n a t i n g seed may  seed.  The  be t e s t e d i n s e v e r a l ways.  pathogenicity  attacking t e s t s with of fungi  on  -  6  -  F i s h e r (12) p l a c e d s u r f a c e s t e r i l i z e d seed i n c o n t a c t w i t h the m y c e l i a l mat o f fungus c u l t u r e s growing i n P e t r i d i s h e s .  To p r o v i d e p r o p e r m o i s t u r e  c o n d i t i o n s f o r the g e r m i n a t i n g seed, r e c t a n g l e s were c u t from t h e agar and the seed was p l a c e d a g a i n s t the c u t s i d e o f the medium. i n o c u l a t e d the g e r m i n a t i n g seed w i t h s p o r e s o f t h e f u n g i .  Hurd (16) One method  used by Rathbun (.25) was t o p l a c e t h e r a d i c l e s o f the g e r m i n a t i n g seed on a b l o c k o f inoculum i n s t e r i l e P e t r i  dishes.  P r e l i m i n a r y t e s t s i n d i c a t e d t h a t , w i t h Douglas f i r seed, the most s u i t a b l e procedure was to p l a c e germinated seed on c u l t u r e s o f the fungus in Petri  dishes.  To o b t a i n the germinates u s e d i n the t e s t s , seed was i z e d and p l a c e d on m a l t agar i n P e t r i d i s h e s . the  surface-steril-  The seeds were l e f t  r a d i c l e had grown a p p r o x i m a t e l y o n e - q u a r t e r i n c h  ( F i g . 3).  until  Seeds  which had n o t been c o m p l e t e l y s u r f a c e - s t e r i l i z e d , as e v i d e n c e d by the growth o f mycelium  from the seed c o a t , were not u s e d i n the t e s t s .  The p a t h o g e n i c i t y o f the v a r i o u s f u n g i was t e s t e d by p l a c i n g a number of  g e r m i n a t e s . a t v a r y i n g d i s t a n c e s from the margin o f the m y c e l i a l mat  (Fig.  4) o r d i r e c t l y on the m y c e l i a l mat ( F i g . 5 ) .  seeds were used w i t h each c u l t u r e .  Ten to twenty-four  As a check on p o s s i b l e i n j u r y t o the  seed due t o t h e s t e r i l i z i n g t r e a t m e n t o r t o d r y i n g o u t i n the P e t r i  dishes,  a number o f germinates were k e p t on s t e r i l e malt agar under the same c o n d i t i o n s as t h e c u l t u r e s . The germinates were l e f t i n the P e t r i d i s h e s f o r a f i v e - d a y p e r i o d . D u r i n g t h i s time those seeds showing e v i d e n c e d by a water-soaked  s i g n s o f h a v i n g been a t t a c k e d , as  appearance  o r d i s c o l o u r a t i o n of the r a d i c l e  were removed and i s o l a t i o n s and i d e n t i f i c a t i o n s were made t o c o n f i r m t h e p a t h o g e n i c i t y o f the fungus  ( F i g s . 6 and 7 ) .  4.  I n o c u l a t i o n of S t r a t i f i e d  7  -  Seed.  To determine whether exposure t o s p o r e s o r mycelium  o f the  contaminat-  i n g f u n g i d u r i n g s t r a t i f i c a t i o n would e f f e c t the g e r m i n a t i o n o f the seed, spores o r mycelium  o f each fungus i s o l a t e d from the seed samples were u s e d  t o c o a t a sample o f t w e n t y - f i v e seeds. s e p a r a t e seed l o t ,  No.  These  seeds were taken from a  5006.  The t r e a t e d seeds were s t o r e d i n a m o i s t chamber a t a temperature 1-2° C. f o r one week and were t h e n p l a c e d on s t e r i l e v e r m i c u l i t e germinated a t room temperature. day p e r i o d was  of  and  The r e c o r d o f g e r m i n a t i o n o v e r a f o u r t e e n  o b t a i n e d and compared to a c o n t r o l l o t o f u n t r e a t e d seed.  Experimental Results 1.  Identifications F i v e hundred and f i f t e e n i s o l a t e s were o b t a i n e d from the t o t a l  o f two  thousand seeds.  and each group was SB 3,  sample  I s o l a t e s w i t h s i m i l a r c h a r a c t e r i s t i c s were grouped  t e n t a t i v e l y i d e n t i f i e d w i t h the n o t a t i o n SB 1, SB  2,  ... e t c . The i d e n t i f i c a t i o n o f the f u n g i , b a s e d on the keys i n Gilman's A Manual  o f S o i l F u n g i (13) i s g i v e n i n the f o l l o w i n g  Isolate SB 1  Identifying  section.  Characteristics  V e g e t a t i v e hyphae s e p t a t e , c o l o u r l e s s , b r a n c h i n g . Conidiophores b e a r i n g p h i a l i d e s i n a s i n g l e  series.  C o n i d i a l heads b l a c k ; v e s i c l e s g l o b o s e , 30-40u i n diameter.  C o n i d i a g l o b o s e , s p i n u l o s e , 3.4-4.3u  i n diameter. A s p e r g i l l u s n i g e r van Tiegham  -  SB 2  8  -  Sporangiophores uribranched, a r i s i n g a t nodes o f a e r i a l mycelium o p p o s i t e r h i z o i d s .  Sporangia  s p h e r i c a l , white a t f i r s t , becoming b l a c k i s h a t m a t u r i t y , a l l o f the same t y p e . hemispherical.  Columellae  Spores round t o o v a l  shaped  10.0-11.5u l o n g x 7 . 0 - 7 . 9 u i n d i a m e t e r . Rhizopus n i g r i c a n s  SB 3  Hyphae s e p t a t e , h y a l i n e . white.  Ehrenberg  M y c e l i a l mat  floccose,  C o n i d i o p h p r e s a r i s i n g from a e r i a l hyphae  hyaline, e l l i p t i c a l , 9.0 x 3.8u. Cephalo sporium c u r t i p e s  SB U  Saecardo  T h i n , c l o s e l y a p p r e s s e d mycelium, s u b f e l t y texture.  M y c e l i a l mat w h i t e .  a p p r o x i m a t e l y one  Slow  growing,  c e n t i m e t e r i n seven  days.  No c o n i d i o p h o r e s o r spores produced i n c u l t u r e , unidentified.  SB 5  Mycelium  mat w h i t e , becoming r o s y - w h i t e .  M i c r o c o n i d i a p r e s e n t ; o n e - c e l l e d , pear  shaped.  M a c r o c o n i d i a s e p t a t e , s i c k l e form, t a p e r i n g a t ends, 3 x 22u.  Chlamydospores smooth, globose,  one c e l l e d , 9-12 x 7-10  u.  Fusarium c o n g l u t i n a n s Wollenweber  SB 6  E x t e n s i v e mycelium w i t h o u t r h i z o i d s ; e r e c t , branched.  sporangiophores  Columellae present, c o l o u r l e s s .  Spores g l o b o s e . Mucor.sp.  Mycelium h y a l i n e , s p a r c e . produced, borne  single.  Abundant  Sclerotia yellowish-  brown i n c o l o u r ; h a r d and g r i t t y , difficulty;  sclerotia  crushing with •  sub-globose, 100-250 x 160-I90u. P e n i c i l l i u m thomii series  Colonies spreading, orange-yellow, with broad zones o f d u l l g r e e n c o n i d i a l heads.  Surface  growth c o n s i s t i n g o f l o o s e l y woven hyphae studded w i t h orange g r a n u l e s : p e r i t h e c i a abundant; reverse yellow-orange.  Gleistothecia yellow,  s p h e r i c a l , 80-100u i n d i a m e t e r .  A s c i 10-12u.  Ascospores l e n t i c u l a r 4.8-5.6 x 3.8-4.4u smooth w a l l e d , e q u a t o r i a l a r e a somewhat f l a t t e n e d , without c r e s t s o r r i d g e s .  C o n i d i a l heads 130u  i n diameter, with c o n i d i a i n chains r a d i a t i n g from a v e s i c l e 25-40u i n d i a m e t e r .  Stalks  smooth, c o l o u r l e s s , 500-1000u i n l e n g t h . P h i a l i d e s i n one s e r i e s 7-10 x 3.4-4.5u. C o n i d i a e l l i p t i c a l t o subglobose,  spinulose,  5.0-6.5u. A s p e r g i l l u s repens (Corda) de Bary  M y c e l i a l mat brownish i n c o l o u r . a r i s e i n groups, unbranched,  Conidiophores  s e p t a t e , n o t con-  s t r i e t e d a t the s e p t a , 75-90u x 5«0-6.5u. Conidia elongated,  egg-shaped or club-shaped  t h r e e t o f i v e c r o s s - w a l l s and  with  several oblique  t r a n s v e r s e w a l l s , brownish, 23 x  lOu.  Macrosporium commune Rabenhorst  Hyphae h y a l i n e , s e p t a t e , b r a n c h e d .  Conidiophores  s h o r t , e r e c t , not d i f f e r e n t i a t e d from t h e mycelium. C o n i d i a dark, o n e - c e l l e d , formed i n l o n g c h a i n s conidiophores,  globose  2.7-3.2u i n  on  diameter.  T o r u l a c o n v u l a t a Harz  Mycelium b r a n c h e d , s e p t a t e , brown, hyphae 2.5-7u i n diameter. septate.  Conidiophores  brown, unbranched,  C o n i d i a brown, curved, borne a t t i p o f  c o n i d i o p h o r e s , w i t h f o u r s e p t a ; 19-45  x 7-14u.  C u r v u l a r i a g e n i c u l a t a ( T r a c y and Boedijn.  M y c e l i a l mat  flat,  colonies broadly  f r u i t i n g a r e a s form as t u f t s , t o green w i t h age; Conidiophores up to 70u  Earle)  spreading,  changing from white  reverse colourless.  a r i s e from a e r i a l mycelium; s e p t a t e ,  i n h e i g h t x 3.0u  t r i c h o t o m o u s l y branched. l O u i n diameter;  i n width,  d i - or  C o n i d i a l heads up  c o n i d i a globose  3.2-3.8u i n  diameter. Trichoderma l i g n o r u m  (Tode) Harz  to  M y c e l i a l mat  t h i n , w r i n k l e d tough, g r a y i s h green.  Reverse deep orange  colour.  Conidiophores erect,  s h o r t , with d i v e r g i n g b r a n c h e s , s i n g l e v e r t i c i l p h i a l i d e s ; p h i a l i d e s few i n v e r t i c i l . smooth, g l o b o s e , 3.2u  of  Conidia  i n diameter.  P e n i c i l l i u m griseum (Sopp) Thorn  C o l o n i e s d u l l green w i t h white a r e a s o f mycelium exposed;  s p r e a d i n g , s p a r s e l y f l o c c o s e a e r i a l hyphae.  Reverse not c o l o u r e d .  C o n i d i o p h o r e s 150-300u x  3.0-3«5u, apex e n l a r g e d t o 5u, b e a r i n g a s i n g l e v e r t i c i l of phialides 9-H  x 2-3u;  penicillus  w i t h c o n i d i a l c h a i n s up t o 300u i n l e n g t h x 15-30u. C o n i d i a p y r i f o r m 3.2-3-5 x 3.6-4.0u; t h i n w a l l e d , verrucose; yellowish-green i n colour. P e n i c i l l i u m spinulosum Thom  C o l o n i e s r e s t r i c t e d i n growth, b l u i s h - g r e e n a t f i r s t , . t u r n i n g greyish. then s o r d i d r e d .  Reverse y e l l o w i s h ,  Conidiophores r i s i n g  c r e e p i n g hyphae 10-35  from  x 2.3-5u; p e n i c i l l u s 20-35u  l o n g , w i t h smooth w a l l s , f i g u r e d v a r i o u s l y from m o n o v e r t i c i l l a t e t o a dense group o f metulae  9-13u  p h i a l i d e s 6-11 eight.  x 2-3u  i n groups o f two  metulae; to f i v e ;  x l.'5-4u. i n v e r t i c i l s o f two t o  C o n i d i a o b l o n g 2-3 x 1.-8-2.5u. P e n i c i l l i u m fellutanum Biourge  - 12 -  Colonies floccose, white at f i r s t , later with a pinkish centre.  Sterile hyphae septate, branched,  hyaline, intermixed with chlamydospore-like segments.  Conidiophores erect 100-200u long, unbranched,  nonseptate; conidial heads 20.0-^26.On in diameter. Conidia globose, 2.3-2.5u i n diameter. Cephalo sporium hum!cola Oudemans Sterile hyphae creeping, spreading, olive-green; hyphae curved, hyaline, branched, 22u thick. Conidia inverted, e l l i p t i c a l , .with.2-3 septa, muriform, verrucose, brown, non-transparent when mature, 17.5-22 x 11-13.5u. Stemphyliua verruculosum Zimmermann Mycelium creeping, whitish, branched, septate. Pycnidia superficial, scattered, without ostiole; dark brown, with setae on a l l sides. Setae of old pycnidia black at lower end, pale olive upward, septate, once to many times dichotomously branched.  Spores broad e l l i p t i c a l ,  biconvex, commonly spiculate at both ends, 5.5-7 x 3 . 5 - 4 u .  Conidiophores dark toward the  base, hyaline above, three times length of spore. Chaetomella horrida Oudemans Mycelial mat whitish, cottony i n texture. Moderate growth rate, approximately four  - 13 -  centimeters i n seven days.  No identifying  conidiophores or spores produced i n culture. Unidentified. SB 20  Mycelial mat white, dense, appre_ssed.  Growth  rate moderate, 7.5 centimeters i n five days. Conidia formed i n acervulus-like structures which develop abundantly on the surface of the mycelium.  Conidia fusiform, 4-septate,  23.1-33 x 7-10u, with guttulate, coloured median cells and hyaline end cells; the two upper median cells darker than the lower. Unidentified.  2.  Incidence and Occurrence of Seed-borne Fungi. The percentage of seed from which fungi were isolated varied with  each seed l o t .  The range for the external isolates was from 20.0 per  cent for seed l o t 4803 to 48.0 per cent for seed l o t 4901 (Table 1 ) . , The  Sa«d Lot N u m b e r of Seed Fungus  TABLE 1 N U M B E R AND P E R C E N T A G E OP ISOLATES O B T A I N E DE X T E R N A L L YP R O B SIX SEED LOTS. 5008 5009 4803 4902 5010 4901 . ^900 . . 150 150 150 150 150 150 Ho. • % No. 2 . 0 6 0.7 2. .0 0 27 2. .3 0 225 U. 13.3 5.3 24 2.0 25 16.6 ,14172.'018.61.3' 3  SB 1 (Aspergillus niger) 2 (Rhizopus nigricans) 3 (Cephalosporlua gurtipes) 4 (unidentified) 5 (Fusarium eonglutiaans) 6 (Mucor ap.) ' • 7 (Penicilllum thomlj) 8 (Aspergillus repens) 9 (Hacrosporiua commune) 10 (Torula convulata) 11 (Cijiyularla geniculate) . 12 (Trichoderma lignorum) \ 13 (Penicilllum grlseum) 14 (P. apinulosum) 15 (P. fallutamim) 16 (Cephaloaporium hum!cola) 17 (Stemphylium verruculosum) 18 '(Cfaaetomella borrida) ' 19 (unidentified) 20 (unidentified) Percentage of seed contaminated  :  26  1 0.1 28 3.1 . 1 0.1  12 1. .0 9 2 11 8 -1.3•  6. .3 0 1  6 .0 1.3  2 0.2 3 0.3 60 ' 6 0.7 3 0.3  6 . 0  -  1 4  -  i n t e r n a l i s o l a t i o n s ranged from 3 . 5 p e r c e n t f o r seed l o t 5 0 1 0 t o 4 3 . 3 per  cent f o r seed l o t  (Table  4902  2 ) .  The p e r c e n t a g e o f seed i n each  tlBU 2  200 -  150 &>  SB  1 2 3 4 5 6 8 9 10 11 12 13  (Aan-rgUlna njprr) ( H h l w a s nigricans) • (CeJnlODjorlm cnrtlpes) (unidentified) ( F p s u i n a conglutlmra) Cincor sp.) (Aspergillus rea-ra) t^fieroraoriun coanine) (Torul*. cormjlata) ( C u r v j l s r l a r»nicnlata) (Trichoderaa llmorum) [ P g d r i l U m ,-rismn)  14 (£-  15 16 17 18 19 20  11 . 22  % -_  1.3 V? '."  • 44  ^ 2.6 2.6  11  -  '. 4 4 i  •  *>.  22 1.0 1.0 13 6.5 6.5 '• 13 11 -  2.6 2.6 1.3  „  11  --  200  150  *  22 u14 22  •  * 1.3 1.3 9.0 9.0 1.3 1.3  v  sg*Tm1r>guaJ  ( £ . f a l l s tamn) (Ccqhalosyorlira buadcala) (SteaohyHua yerrncclosan) tQJeatqaella hurt Ida) (mOaentlfled) (tnrUertirtM)  Ho.  16 10.5 10.5 16  150 Bo.  6 6 3.0 3.0 12 ••6 6.0 12 .0 1.0 22 1.0 3 1.5 3 1.5 11 -- 16 8.0 8.0 16  .  .4 .4 10 10 22  3.0 3.0 6.7 6.7 1.3 1.3  . 2 2 *4  1.3 1.3 3.0 3.0  11 11  1 1  -  3  1.5  22 6 3 5 6 9  1.3 1.3 4.0 2.0 3.3 4-0 6.0  11 11 10 2 6 3 3  -  11 10 10  _  5.0  _ _ 6.7 6.7  11  -  11  -  -  2  j ^  8  1.0 ' 3.0 1.5 ., 1.5 .<  15 1.3 51 4 . C 11 1.0 j Q^J 3 3)3 41 3 7  44 22.0 .0  • 1 1  • '200 Bo. %  2  5.3  8 3  5.3 2.0  1  t.O , /  3 ^ 1 1  0.3 0  l i : i  0  2.0  ^ 2  0.2  24  2.2  5  0.5  5  -  0  1  " .» j't 'g  17 21 3  0.; 1.6  2.0 0.3  seed l o t from which a t l e a s t one i s o l a t e was o b t a i n e d i s g i v e n i n T a b l e 3 .  TABLE 3  PERCENTAGE OF SEED IN EACH SEED LOT FROM WHICH AT LEAST 01IE ISOLATE WAS OBTAINED  '  Internal  External  r  Isolations  Seed  Percentage  Lot  Isolations  No. of  No. of  Percentage Seed Examined  Seed Sxamined  9,2  150  20.0  150  150  48.0  200  11.5  4902  150  26,6  150  43.3  5008  150  36.6  200 -  32.0  5009  150  24.6 •  150  34.7  150 •  33.3  200  3.5  4803 4901  5C10 51GT  :  '  .50.  - '-  12.0  -  15  -  The f u n g i d i d n o t o c c u r c o n s i s t e n t l y i n a l l seed l o t s and t h e p e r c e n t ( T a b l e s 1 and 2 ) .  age o f each fungus i n t h e d i f f e r e n t seed l o t s a l s o v a r i e d  The most c o n s i s t e n t l y i s o l a t e d fungus was Rhizopus n i g r i c a n s which was i s o l a t e d from t h e seed c o a t o f a l l s i x seed l o t s and i n t e r n a l l y from a l l b u t seed l o t 5010 and 51GT. Some o f t h e f u n g i , e.g., P e n i c i l l i u m t h o m i i . Macrosporium Stemphylium  (SB 4 and SB 20)  v e r r u c u l o s u m . and two u n i d e n t i f i e d f u n g i  were i s o l a t e d from o n l y one seed l o t ,  commune»  and o t h e r s , e.g. P e n i c i l l i u m t h o m i i  were i s o l a t e d o n l y from t h e seed c o a t o r o n l y from s u r f a c e - s t e r i l i z e d  seed.  The l a t t e r were A s p e r g i l l u s r e p e n s . Macrosporium  commune. P e n i c i l l i u m  f e l l u t a n u m . Cephalosporium h u m i c o l a . Stemphylium  verruculosum.  h o r r i d a , and two u n i d e n t i f i e d f u n g i  (SB 19 and SB 2 0 ) .  Chaetomella  The o c c u r r e n c e o f  the f u n g i i n t h e v a r i o u s seed l o t s i s g i v e n i n T a b l e 4-  TABLE 4. OCCQRREHCe Of FUNGI IN HIS DIFFERENT SEED LOTS. EXTERNAL ..AND INTERNAL ISOLATIONS ARE INDICATED.  Seed Lot  4803 ,  x  ex. i n .  Occurrence Fungus  4901  4902  ex. i n .  ex. i n .  5008 ex. In.  .'•  Aspergillus nicer A. repens Cephalosporium curtlpes C. humicola Chaetomella horrida Curvulfcrja genlculata Fusarium corulut loans Macroscoriua commune '.'ucor sp. ?°nlcillluin fellutanum £. rrlseum ?. thomii • Rhizopus nif-.rlcans Stemohyllum verruculosum Torul& comrulata Trichoderma lignorum £B 4 (unidentified) £B 19. (unidentified) '' SB £0 (unidentified)  X  X  X  X  X  X  X X X X  x X  X  .X  X  X  X  .*  *  ' X X  X  X X X X X.  X X'  X  X  X X  X X . X  X 'X " X X ' X X  5009  5010  51GT  ex. i n .  ex. i n .  ex. i n .  - 16 3.  Pathogenicity Tests The o c c u r r e n c e o f t h e p a t h o g e n i c f u n g i i s shown i n T a b l e 5.  TABLE 5 OCCURRENCE OF THE PATHOGENIC FDBCI IN THE SEVEN SEED LOTS.  Seed l o t Seeds Examined  4803  4901  4902  5008  5009  300  350  300  350  300  350  50  12  7 6  8 2 1  5 8  1 5 1  2  2 1  3  5010  510T  Funeus Aspergillus repens Cephalosporiua curtioes Chaetoaiella horrida Fusarium comrlutinans Penicillium fellutanma grlseuo £. snlnulosum Etemohvlium verruculosum Trichoderma llgnorum  4  infected  "i of seed • Germination values •  .  3  2  3  10 3  8  1  19 3  5 14 9  10 6  22  10  4  39  47  u  49  20  1.3  11.1  15.6  11.7  16.3*  37*  40*  43*  50?  SB 19 (urddentifiedT  Total  6  34*  3  1  6  5.7  12.0  30*  65*  The r e s u l t s o f t h e p a t h o g e n i c i t y t e s t s a r e summarized i n T a b l e  6.  TABUS 6 RESULTS OF PATHOGENICITY TESTS. AT THE END OF A FIVE DAT PERIOD  Fungus  AsDerelllus reoens CephalosBorium cuytiDesChaetomella horrida Fusarium conrlutlnans P e n l c l l l l u a fellutanum P. rriseun P. SBinulosum SteDDhylium verruculosum Trichoderma llgnorum. SB 19 (unidentified)  o  Number of Radicles Exposed.  11  Radicles attacked ......  4 v . .  .  . Number . 7  16  23  29 11 15  20  35  26  11 ii  ll  24  11 9  -ro-  9.. -•  10 '  24 21  Percentage : 63.6 69.5 69.0 100.0 60.0 74.3 81.7 90.9 . 72.6 . 87.5  - 17 -  These t e s t s showed t h a t t h e f o l l o w i n g f u n g i were c a p a b l e o f a t t a c k i n g t h e r a d i c l e s A s p e r g i l l u s r e p e n s , Cephalosporium  c u r t i p e s . Chaetomella h o r r i d a .  Fusarium c o n g l u t i n a n s . P e n i c i l l i u m f e l l u t a n u m . _P. griseum. P. spinulosum, Stemphylium v e r r u c u l o s u m , Trichoderma l i g n o r u m , and SB 19. namely, Cephalosporium  F i v e o f these  c u r t i p e s . Fusarium c o n g l u t i n a n s . P e n i c i l l i u m  griseum. P. spinulosum and Trichoderma l i g n o r u m were i s o l a t e d from the seed c o a t a s w e l l as from w i t h i n the seed.  The o t h e r f i v e f u n g i namely  A s p e r g i l l u s repens, Chaetomella h o r r i d a , P e n i c i l l i u m f e l l u t a n u m , Stemp h y l i u m v e r r u c u l o s u m and SB 19 were i s o l a t e d o n l y from w i t h i n the seed. The r e m a i n i n g t e n f u n g i namely A s p e r g i l l u s n i g e r . h u m i c o l a . C u r v u l a r i a g e n i c u l a t a . Macrosporium  Cephalosporium  commune. Mucor sp., P e n i c i l -  l i u m t h o m i i . Rhizopus n i g r i c a n s . T o r u l a c o n v u l a t a , SB 4 and SB 20, d i d n o t a t t a c k the r a d i c l e s o f the g e r m i n a t i n g seed.  Of t h e s e , A s p e r g i l l u s  niger.  C u r v u l a r i a g e n i c u l a t a , Mucor sp., Rhizopus n i g r i c a n s , T o r u l a c o n v u l a t a , and SB 4 were i s o l a t e d from the seed c o a t and from w i t h i n t h e seed. P e n i c i l l i u m t h o m i i was i s o l a t e d o n l y o f f t h e seed c o a t and Cephalosporium h u m i c o l a , Macrosporium sterilized  4.  commune, and SB .20 were i s o l a t e d o n l y from  surface  seed.  I n o c u l a t i o n o f S t r a t i f i e d Seed. The percentage g e r m i n a t i o n o f t h e seeds t r e a t e d w i t h spores o r mycelium  o f the d i f f e r e n t f u n g i d u r i n g s t r a t i f i c a t i o n i s g i v e n i n T a b l e 7.  Under  the c o n d i t i o n s o f t h i s experiment t h e r e was a r e d u c t i o n i n g e r m i n a t i o n , compared t o t h e c o n t r o l o f u n t r e a t e d seed (65 p e r cent g e r m i n a t i o n ) , w i t h f i v e o f the f u n g i .  These f u n g i were A s p e r g i l l u s n i g e r ,  Cephalosporium  c u r t i p e s . Fusarium c o n g l u t i n a n s . Mucor s p . and P e n i c i l l i u m t h o m i i . seven o t h e r f u n g i Cephalosporium h u m i c o l a . Chaetomella h o r r i d a ,  With  Curvularia  - 18 -  TABLE 7. RESULTS OF GERMINATION TESTS WITH STORE-INOCULATED SEED.  Percentage Germination (14 days)  Number o f seeds treated  Fungus  Aspergillus niger A. repens Cephalosporium curtipes _C. humicola Chaetomella horrida Curvularia geniculata Fusarium conglutinans Macrosporium commune Mucor spi Penicillium fellutanum P. griseum P. spinulosum P. thomii Bhlzop'is nigricans Stemohyllum verruculosum Torula convulata Trichoderma lignorum SB 4 (unidentified) SB 19 (unidentified) SB 20 (unidentified)  H  ' ;  •  Control (untreated)  '  25 . _ -. -.si*..'*'•-  60 .." 76 ~ 61* 67 66 66 64. 84. 56* 76 66 91 43* 68 80 79 68 68 77  "  ,80  65  lower than the control  g e n i c u l a t a . P e n i c i l l i u m griseum. Rhizopus n i g r i c a n s . Trichoderma l i g n o r u m . and SB L t h e percentage g e r m i n a t i o n was l e s s t h a n seventy p e r c e n t . S i n c e the seed used f o r the s t r a t i f i c a t i o n t e s t s had a h i g h  germin-  a t i o n v a l u e (80 p e r cent) when t e s t e d p r e v i o u s l y , i t i s f e l t t h a t t h e lower v a l u e o b t a i n e d b y the c o n t r o l i n the experiment i s n o t a t r u e of  indication  t h e g e r m i n a t i o n c a p a c i t y o f t h i s seed and t h e r e f o r e the h i g h e r v a l u e  s h o u l d be used when comparing i s a l s o u s e d i n comparing as shown i n T a b l e 8.  t h e r e s u l t s o f t h i s experiment.  This value  the p a t h o g e n i c and i n h i b i t o r y e f f e c t o f t h e f u n g i  -  19  -  TABLE S StiaiABI OF THE PATHOGEHICITI AND INHIBITING EFFECT OF THE ISOLATED FDBGI.  Effect on seed  Fungus Pathogenic  Honpathogenic  Reduced* germination X  Aspergillus niger, A. reoena r.^phglasporlua curtioes £. humlcola nhr,Btonglla horrida j Curvularla pellicula ta I Fusarium conglutinans Jacrosporlum commune I Mucor"BP. I £eiilclUlna^f^u.tanm _P. griseum i £ . spinulosum P. thomll Rhizopus nigricans fir.«mphyllMi vgrruculosum • Torula coovulate • Ifichjoderma. llcnorum .' SB 4 SB 19 SB 20  X X X  R  X X x x  X X  X  1  X  x redaction below 65 per cent - reduction below 80 per cent_  Discussion  Seed g e r m i n a t i o n may be i n f l u e n c e d b y t h e n a t u r e o f t h e seed and b y t h e t r e a t m e n t  itself  i t r e c e i v e s d u r i n g c o l l e c t i o n , e x t r a c t i o n and s t o r a g e .  Those seeds which r i p e n i n t h e s p r i n g w i l l germinate  i n a v e r y s h o r t time  under f a v o u r a b l e c o n d i t i o n s whereas t h o s e which r i p e n i n t h e f a l l ,  when  n a t u r a l c o n d i t i o n s f o r g e r m i n a t i o n a r e l e s s f a v o u r a b l e , have a dormant stage which can be b r o k e n o n l y b y exposure t o c o l d f o r a p e r i o d o f t i m e . The degree o f m a t u r i t y o f seed h a s a marked i n f l u e n c e o n t h e germination.  Seed which i s immature g e n e r a l l y h a s a lower and more i r r e g u l a r  g e r m i n a t i o n t h a n r i p e seeds (2, ,20). may be a r e s u l t o f adverse which p r e v e n t e d  The f a i l u r e o f seed t o mature f u l l y  c l i m a t i c c o n d i t i o n s d u r i n g t h e growing  season  t h e seed from r e a c h i n g m a t u r i t y o r , i n t h e case o f c o n i f e r o u s  s p e c i e s , o f premature c o l l e c t i o n o f t h e cones b e f o r e t h e seed h a d r i p e n e d .  - 20  -  When seed i s p r o v i d e d w i t h s u i t a b l e m o i s t u r e and temperature c o n d i t i o n s the g e r m i n a t i o n p r o c e s s w i l l b e g i n and w i l l c o n t i n u e u n t i l t h e embryo has developed i n t o a s e e d l i n g .  Under normal  c o n d i t i o n s the s e e d l i n g w i l l  e s t a b l i s h e d b e f o r e t h e f o o d s u p p l y s t o r e d i n the seed i s exhausted. ever, under f l u c t u a t i n g temperature  be How-  and m o i s t u r e c o n d i t i o n s the seeds a r e  s t i m u l a t e d t o b e g i n g e r m i n a t i o n which may  n o t t h e n be completed.  Since  a p o r t i o n o f the f o o d r e s e r v e i s u s e d each time g e r m i n a t i o n s t a r t s , r e p e a t e d s t i m u l a t i o n w i l l cause the f o o d s u p p l y t o become exhausted the r e s u l t t h a t g e r m i n a t i o n may  with  be i n c o m p l e t e o r even f a i l under f a v o u r a b l e  conditions. Premature s t i m u l a t i o n o f t h e seed may be p r e v e n t e d by s t o r i n g the seed i n c o o l , d r y l o c a t i o n s it  i s used.  (6, p.52)  from the time i t i s c o l l e c t e d  until  The optimum s t o r a g e c o n d i t i o n s f o r c l e a n e d seed a r e a t  temperatures r a n g i n g from 35° - 40°F. o r even below f r e e z i n g and a low m o i s t u r e c o n t e n t (7, 17,  temperatures  23).  A l t h o u g h f l u c t u a t i n g c o n d i t i o n s w i l l reduce the v i t a l i t y o f seed, h i g h temperatures  (up t o 170°F.) can be endured d u r i n g the  extraction  process-, p r o v i d e d the r e l a t i v e h u m i d i t y o f the k i l n and the seed's c o n t e n t a r e low to  (27).  Temperatures  moisture  i n excess o f t h i s however a r e h a r m f u l  the seed and must be a v o i d e d i f the seed i s n o t t o be damaged. M e c h a n i c a l i n j u r y may  de-winging p r o c e s s .  be caused to the seed d u r i n g the e x t r a c t i o n and  Most commercial  e x t r a c t i o n p r o c e s s e s tumble the  i n drums which p e r m i t the seed to f a l l  out where i t can be  cones  collected.  While i n the drums the l o o s e seed i s s t r u c k by the cones and o t h e r d e b r i s and as a r e s u l t o f t h i s rough t r e a t m e n t some o f t h e seed c o a t s w i l l broken. treatment.  A s i m i l a r type o f damage may T h i s t y p e o f i n j u r y may  a l s o be caused by the  be  de-winging  n o t be a p p a r e n t from i n s p e c t i o n o f the  seed b u t may  appear upon g e r m i n a t i o n through the abnormal s p l i t t i n g o f the  seed c o a t ( l l ) .  Seeds which have been damaged i n t h i s way  v i g o r o u s and more s u s c e p t i b l e t o d e t e r i o r a t i o n The dormancy o f seeds may medium a t a low temperature  are l e s s  (3).  be b r o k e n by p l a c i n g them i n a m o i s t  f o r a p e r i o d o f s e v e r a l weeks.  During t h i s  p e r i o d o f " s t r a t i f i c a t i o n " chemical changes concerned w i t h the c o n v e r s i o n o f f o o d r e s e r v e s from the i n s o l u b l e t o s o l u b l e s t a t e take p l a c e w i t h i n the embryo and endosperm. germinate  These changes c o n d i t i o n t h e seeds so t h a t they  almost immediately when moved to a h i g h e r  temperature.  Some o f the t y p i c a l changes which take p l a c e d u r i n g t h i s as summarized by Baldwin  (6, p . 1 1 3 ) ,  treatment,  are:  1.  I n c r e a s e i n amount o f water  absorbed.  2.  Increase i n t o t a l  3.  I n c r e a s e o f s u c r o s e , r e d u c i n g s u g a r s , amino n i t r o g e n , amino  acidity.  acids. 4.  Increased r e s p i r a t i o n .  5.  Appearance o f c a t a l a s e , o x i d a s e and p e r o x i d a s e a c t i v i t y .  I n mature, whole seed t h e s e p r o c e s s e s c o n t i n u e i n an o r d e r l y manner u n t i l g e r m i n a t i o n i s complete b u t when the seed i s immature i t i s p o s s i b l e t h a t g e r m i n a t i o n i s p r e v e n t e d by an i n h i b i t i n g substance p r e s e n t i n the endosperm (19)•  The f a i l u r e of damaged seed t o germinate  i s probably  due to t h e f a c t t h a t the i n j u r y t o t h e seed c o a t has p e r m i t t e d t h e o x i d a t i o n o f m a t e r i a l p r i o r t o s t r a t i f i c a t i o n and because o f t h i s , as w i t h poor s t o r a g e c o n d i t i o n s , the seed i s u n a b l e to complete  i t s germination.  I n a d d i t i o n t o t h e f a c t o r s d i s c u s s e d above, which would i n f l u e n c e the g e r m i n a t i o n percentage o f seed which had been exposed t o t h e s e  con-  d i t i o n s , the presence of f u n g i c o u l d a l s o a f f e c t the g e r m i n a t i o n p r o c e s s .  - 22 The e f f e c t produced b y the f u n g i c o u l d r e s u l t from t h e a n t i b i o t i c p r o p e r t i e s o f t h e i r m e t a b o l i c p r o d u c t s ; i t has been shown t h a t t h e s e compounds c a n i n h i b i t g e r m i n a t i o n and s e e d l i n g growth a t low c o n c e n t r a t i o n s (8).  A l t h o u g h n o t a l l f u n g i produce  t h e s e a n t i b i o t i c substances and t h e  amounts produced b y a s m a l l mass o f mycelium would be v e r y l i m i t e d ,  there  i s t h e p o s s i b i l i t y t h a t the p r o d u c t i o n o f such m a t e r i a l s b y t h e f u n g i  con-  t a m i n a t i n g t h e seed would i n f l u e n c e g e r m i n a t i o n under t h e c o n d i t i o n s imposed d u r i n g s t r a t i f i c a t i o n and g e r m i n a t i o n t e s t s .  B e s i d e s the i n h i b i t -  i n g e f f e c t , t h e p a r a s i t i c a c t i o n o f t h e f u n g i would a l s o reduce g e r m i n a t i o n . T h i s a c t i o n would be most e f f e c t i v e a g a i n s t seed which h a d b e e n p r e v i o u s l y weakened o r damaged f o r t h e f u n g i would be a b l e t o p e n e t r a t e t h i s very e a s i l y .  seed  Where the f u n g a l c o n t a m i n a t i o n i s e x t e r n a l t h e development  o f t h e f u n g i may be c o n t r o l l e d b y t h e use o f a f u n g i c i d e  ( 1 , 4) b u t where  the f u n g i have p e n e t r a t e d t h e seed t h i s treatment i s i n e f f e c t i v e and l o s s e s from t h i s type o f c o n t a m i n a t i o n c o u l d n o t be p r e v e n t e d . In other i n v e s t i g a t i o n s  (12, 14, .26, 28, 30) i t was f o u n d t h a t t h e f u n g i  i s o l a t e d from t h e seed c o u l d be grouped a c c o r d i n g t o t h e i r p a t h o g e n i c Some were found t o be p a t h o g e n i c whereas o t h e r s i n h i b i t e d  ability.  germination b u t  d i d n o t a t t a c k t h e seed, and o t h e r s were n e i t h e r p a t h o g e n i c n o r i n h i b i t o r y . A s i m i l a r d i v i s i o n c o u l d be made o f t h e f u n g i i s o l a t e d i n t h i s Under t h e c o n d i t i o n s o f t h e s e experiments  study.  f i v e o f t h e f u n g i were p a t h o g e n i c  b u t d i d n o t i n h i b i t t h e g e r m i n a t i o n o f s t r a t i f i e d seed. p a t h o g e n i c f u n g i and seven o f t h e non-pathogenic  F i v e other  fungi inhibited  seed  g e r m i n a t i o n and t h r e e f u n g i were n o t p a t h o g e n i c n o r d i d they i n h i b i t seed germination. S i n c e b o t h p a t h o g e n i c and i n h i b i t i n g f u n g i were found i n t h e v a r i o u s seed l o t s , t h e i r presence c o u l d have h a d a n adverse e f f e c t on t h e germin-  - 23 -  a t i o n v a l u e s o f these seed.  However s i n c e the c o n d i t i o n o f the seed  i t s t r e a t m e n t p r i o r t o use a l s o i n f l u e n c e the g e r m i n a t i o n , t h e low  and  germin-  a t i o n percentage o f the v a r i o u s seed l o t s examined were p r o b a b l y due  to  the combined e f f e c t s o f these t r e a t m e n t s and the a c t i v i t y o f the contaminating  fungi.  Summary and C o n c l u s i o n s .  I n t h i s study a number o f f u n g i were i s o l a t e d from d i f f e r e n t l o t s o f Douglas f i r s e e d .  Some o f these f u n g i were c a r r i e d on the seed c o a t and  o t h e r s were c a r r i e d w i t h i n the seed. Although the f u n g i i s o l a t e d do n o t l i k e l y r e p r e s e n t the  complete  fungus f l o r a o f Douglas f i r seed, a number o f the genera r e p r e s e n t e d c o r respond to t h o s e i s o l a t e d from o t h e r s p e c i e s o f c o n i f e r o u s seed by o t h e r investigators.  I n t h e s e i n v e s t i g a t i o n s the f u n g i c o u l d be grouped  on the  b a s i s o f t h e i r p a t h o g e n i c i t y and the f u n g i i s o l a t e d i n t h i s s t u d y c o u l d be s e p a r a t e d on a s i m i l a r b a s i s .  Under the c o n d i t i o n s o f t h e s e  experiments  f i v e o f the f u n g i were p a t h o g e n i c b u t d i d n o t i n h i b i t the g e r m i n a t i o n o f s t r a t i f i e d seed.  F i v e o t h e r p a t h o g e n i c f u n g i and  seven o f the non-  p a t h o g e n i c f u n g i i n h i b i t e d seed g e r m i n a t i o n and t h r e e f u n g i were n e i t h e r .pathogenic nor Exposure  inhibitory. t o m o i s t u r e and f l u c t u a t i n g temperatures  due to  improper  s t o r a g e and m e c h a n i c a l i n j u r y a r e f a c t o r s which a f f e c t the v i a b i l i t y seed.  The a b i l i t y o f seed t o germinate may  of  a l s o be a f f e c t e d b y the  p a r a s i t i c a c t i o n o f f u n g i and by a n t i b i o t i c substances produced by A l t h o u g h i t i s not p o s s i b l e t o determine which treatment o r might have a f f e c t e d an i n d i v i d u a l seed l o t , i t i s l i k e l y t h a t the  fungi. condition low  - 24 -  germination of the several seed lots examined was a result of exposure of the seed to some or a l l of these conditions at various stages i n i t s collection, processing and handling.  Bibliography. 1. Allen, G. S. Mold free germination of coniferous seed. 1947.  Journ. For. 45: 51  2. Allen, G. S. Factors affecting the. v i a b i l i t y and germination behaviour of coniferous seed. I I . Cone and seed maturity, Pseudotsuga menziesii (Mirb.) Franco. For. Chron. 34; 275-282. 1958. 3.  Allen, G. S. Factors affecting the v i a b i l i t y and germination behaviour of coniferous seed. I I I . Commercial processing and treatments similar to processing, Pseudotsuga menziesii (Mirb.) Franco, and other species. For. Chron. 34: 283-298. 1958.  4. Allen, G. S. and W. Bientjes. Studies on coniferous tree seed at the University of British Columbia. For. Chron. 30: 183-196. 1954. 5. Andrus, C. F. Seed transmission of Macrophomina phaseoli,  633. 1938.  Phytopath. 28: 621-  6. Baldwin, H. I. Forest Tree Seed.  Chronica Botanica Co. Waltham, Mass. 1942.  7. Barton, L. V. Storage of some coniferous seed. 7: 379-404. 1935.  Contrib. Boyce Thompson Inst.  8. Brian, P. W. Effects of antibiotics on plants. (pp.414-417). 1957).  Annual Review of Plant Phys. 8  - 25 9. Chilton, St. J . P. Some pathogenic fungi occurring i n the seed of red and subterranean clover. Phytopath. 32: 738-739. 1942. •  10. Crozier, W. Materials and methods i n controlling seed-contaminating microorganisms. Proc. Assoc. Off. Seed Anal. N. Amer. (1942) 1943. 11.  Eliason, E. J. and C. E. Heit. The results of laboratory tests as applied to large scale extraction of red pine seed. Journ. For. 38: 426-429. 1940.  12.  Fisher, P. L. Germination reduction and residual decay of conifers caused by certain fungi. Journ. Agric. Res. 62: 87-95. 1941.  13.  Gilman, J . C. A manual of s o i l fungi. 392 pp.  14.  Hartley, C , T. C. Merrill and A. S. Rhoads. Seedling diseases of conifers. 1918.  15.  Iowa State College Press. 1945.  Journ. Agric. Res. 15: 521-558.  Heit, C. E. and E. J. Eliason. Coniferous tree seed testing and factors affecting germination and seed quality. N.Y. State Agric. Expt. Sta. Techn. Bull.255. 1940.  16.  Hurd, Annie, M. Seed coat injury and v i a b i l i t y of seeds of wheat.and barley as factors i n susceptibility to molds and fungicides. Journ. Agric. Res. 21: 99-122. 1921.  17.  Isaac, L. A. Cold storage prolongs the l i f e of noble f i r seed and apparently ' increases germination. Ecology 15: 216-217. 1934.  18.  Koehler, B. Natural mode of entrance of fungi into corn ears and some symptoms that indicate infection. Journ. Agric.-Res. 64: 421442. 1942.  - 26 -  19.  Koshimizu, J . On the r e l a t i o n between t h e r i p e n i n g s t a g e s o f the maize-seed and i t s g e r m i n a t i o n . B o t . Mag. (Tokyo), 50: 504-513. 1936.  20.  Maki, J . E . S i g n i f i c a n c e and a p p l i c a b i l i t y o f seed m a t u r i t y i n d i c e s f o r ponderosa p i n e . J o u r n . F o r . 38: 55-60. 1940.  21.  M e t c a l f , W. F u m i g a t i n g and s t e r i l i z i n g t r e e seed. 512. 1925.  22.  J o u r n . F o r . 23: 508-  Muskett, A. E . and J . P. Malone. The U l s t e r method f o r the examination o f f l a x seed f o r the presence o f seed-borne p a r a s i t e s . Ann. A p p l . B i o l . 28: 8. 1941.  23.  Nelson, M. L . S u c c e s s f u l s t o r a g e o f s o u t h e r n p i n e seed f o r seven y e a r s . J o u r n . F o r . 38: 443-444. 1940.  24.  Nobles, M i l d r e d , K. Studies i n f o r e s t pathology. VI. I d e n t i f i c a t i o n of cultures of wood-rotting f u n g i . Can. J o u r n . Res. C. 26: 281-431. 1948.  25.  Rathbun, Annie, E . Methods o f d i r e c t i n o c u l a t i o n w i t h damping-off P h y t o p a t h 11: 80-94. 1921.  26.  Rathbun-Gravatt,  fungi.  Annie, E .  G e r m i n a t i o n l o s s o f c o n i f e r seeds due t o p a r a s i t e s . A g r i c . Res. 42: 71-92. 1931. 27.  Journ.  R i e t z , R. C. and K. E. K i m b a l l . K i l n s c h e d u l e s f o r e x t r a c t i n g r e d p i n e seed from f r e s h and s t o r e d cones. J o u r n . F o r . 38: 430-434. 1940.  28.  Roth, L . F. and A. J . R i k e r . I n f l u e n c e o f temperature, m o i s t u r e and s o i l r e a c t i o n on dampingof f . J o u r n . A g r i c . Res. 67: 273-293. 1943.  -27  29.  Tervet,  I.  -  W.  The i n f l u e n c e o f f u n g i on s t o r a g e , on seed v i a b i l i t y , and l i n g v i g o r of soybeans. P h y t o p a t h . 35: 3-15. 1945. 30.  Tint,  H. Virulence  31.  seed-  T o o l e , E . H. The  293.  and  o f F u s a r i a on P e a r l L.  conifers.  35: 421-439.  Drummond.  germination of cotton  1924.  Phytopath.  seed.  J o i i r n . A g r i c . Res.  28:  285-  1945  PLATE I  F i g u r e 1. Method o f p l a n t i n g the s e e d t o i s o l a t e t h e . fungi. Douglas f i r seed on m a l t agar i n a Petri dish. F i g u r e ,2. Growth o f f u n g i from seed p l a c e d on malt agar. F i g u r e 3. S u r f a c e s t e r i l i z e d agar.  seed g e r m i n a t i n g on m a l t  F i g u r e s 4 and 5. Methods o f t e s t i n g the p a t h o g e n i c i t y o f the f u n g i . F i g . 4. S u r f a c e s t e r i l i z e d germinates p l a c e d a t v a r y i n g d i s t a n c e s from the edge, o f a m y c e l i a l mat o f a fungus. F i g . 5. S u r f a c e s t e r i l i z e d germinates on a m y c e l i a l mat. F i g u r e 6. Germinates whose r a d i c l e s have been a t t a c k e d by a p a t h o g e n i c f u n g u s . F i g u r e 7. Germinates on the m y c e l i a l mat o f a nonpathogenic fungus. The r a d i c l e s have not been a t t a c k e d by the fungus.  

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