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Electron microscope study of a mycelial mutant of Ustilago hordei (Pers.) Lagerh. Stein, Carla Winnifred 1970

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AN ELECTRON MICROSCOPE STUDY OF A MYCELIAL MUTANT OF USTILAGO HORDE I (PERS.) LAGERH. by CARLA WINN I FRED STEIN B.A., Hons. B i o l . U n i v e r s i t y o f Saskatchewan R e g i n a , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SC I ENCE i n the Department o f Botany We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA F e b r u a r y , 1970. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of TtfWATCTY  The University of British Columbia Vancouver 8, Canada D a t e March 13, 1970 ABSTRACT A mutant m y c e l i a l c o l o n y o f List i 1 ago horde i ( P e r s . ) . Lagerh.' was s t u d i e d by l i g h t and e l e c t r o n m i c r o s c o p y t o compare i t s g e n e r a l morphology w i t h r e s u l t s o b t a i n e d from e a r l i e r c y t o l o g i c a l s t u d i e s . I t s u l t r a s t r u c t u r e i s a l s o compared w i t h t h a t o f o t h e r f u n g i . The r e s u l t s o b t a i n e d from l i g h t m i c r o s c o p y g e n e r a l l y a g r e e w i t h t h o s e from e a r l i e r work. C e l l s o f the m y c e l i a a r e v e r y l o n g , i r r e g u l a r i n l e n g t h , and c o n t a i n numerous m i t o c h o n d r i a and one t o s e v e r a l n u c l e i . E l e c t r o n m i c r o s c o p y r e v e a l s the w a l l s o f the m y c e l i a l c e l l s t o be s i n g l e l a y e r e d f i b r i l l a r s t r u c t u r e s . In a p i c a l r e g i o n s a l l the c h a r a c t e r i s t i c o r g a n e l l e s a r e p r e s e n t . The e l o n g a t e m i t o c h o n d r i a have p l a t e - l i k e c r i s t a e t h a t l i e p a r a l l e l t o the long a x i s o f the m i t o c h o n d r i and the o u t e r bounding membrane i s o f t e n t h i c k e r than the i n n e r one; t h e s e two f e a t u r e s o f t h e m i t o c h o n d r i a a r e both u n u s u a l . E n d o p l a s m i c r e t i c u l u m i s s p a r s e . Branch hyphae and s e p t a e a r e not p r e s e n t . In d i f f e r e n t i a t e d a r e a s t h e r e i s an i n c r e a s e i n the number o f v e s i c l e s and l a r g e r v a c u o l e s . M i t o c h o n d r i a undergo r a t h e r u n i q u e changes whereby c o n c e n t r i c l a m e l l a e a r e formed i n a b u l g e o f o u t e r m i t o c h o n d r i a l membrane. These c o n c e n t r i c l a m e l l a e a r e e n g u l f e d by v a c u o l e s i n w h i c h the l a m e l l a e appear t o be t r a n s f o r m e d i n t o t u b u l a r b o d i e s . A t some p o i n t t h e s e v a c u o l e s f u s e w i t h the plasma membrane and r e l e a s e t h e i r c o n t e n t s i n t o t he space between t h e plasma membrane and t h e c e l l w a l l . The v a c u o l e s seem t o a c t as lysosomes. Branch hyphae and s e p t a e a r e p r e s e n t . Degenerate r e g i o n s a r e v e r y v a c u o l a t e d and c o n t a i n many membranous s t r u c t u r e s . Few o r g a n e l l e s a r e p r e s e n t . M i t o c h o n d r i a a r e d i s t o r t e d and t h e i r c r i s t a e s w o l l e n o r a b s e n t . Dictyosomes and lomasomes a r e not p r e s e n t t h r o u g h o u t t h e hyphae. TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 5 I . Mater i a 1 . . . 5 I I . M a t i n g T e s t . 5 I I I . L i g h t M i c r o s c o p y • 5 IV. P r e p a r a t i o n f o r E l e c t r o n M i c r o s c o p y . . . . . . . 6 RESULTS. 8 I. M a t i n g T e s t . . . . '.. . . • • 8 I I . L i g h t M i c r o s c o p y 8 I I I . E l e c t r o n M i c r o s c o p y . . 8 A. A p i c a 1 . r e g i o n s o f hyphae 9 B. D i f f e r e n t i a t e d r e g i o n s o f hyphae ]h C. Degenerated r e g i o n s o f hyphae . . . . . . . . 17 DISCUSSION. . . . . . . . . . . . 19 SUMMARY. 36 BIBLIOGRAPHY . . . . . . . . . . 38 APPENDIX A. . . . . . . . . . h7 APPENDIX B. . . . . . . . 9^ APPENDIX C . • . • • • • . . . 50 APPENDIX D 51 TABLE OF PLATES P l a t e 1. L i g h t m i c r o g r a p h s showing the hyphae o f the m y c e l i a l mutant of Ust i1 ago horde i . P l a t e s 2-5- U l t r a s t r u c t u r e o f the a p i c a l r e g i o n s o f Ust i1 ago horde i hyphae. P l a t e s 6-11. U l t r a s t r u c t u r e o f t h e d i f f e r e n t i a t e d r e g i o n s o f Ust i1 ago h o r d e i hyphae. P l a t e s 12-15. U l t r a s t r u c t u r e o f the s e p t a and d e g e n e r a t e d r e g i o n s o f Ust i1 ago h o r d e i hyphae. LEGENDS FOR SYMBOL B . = b r a n c h C = c o n c e n t r i c l a m e l l a e ER = e n d o p l a s m i c r e t i c u l u m F = f l o c c u l e n t c y t o p l a s m G = g l y c o g e n g = m i t o c h o n d r i a l g r a n u l e s i = u n i d e n t i f i e d body L = l i p i d M = m i t o c h o n d r i o n ML = membrane l a m e l l a e complex MT = m i c ro tubu1es N = n u c l e u s NE = n u c l e a r e n v e l o p e NP = n u c l e a r pore NU = n u c l e o l u s 0 = e l e c t r o n opaque a r e a P = p r o j e c t i o n PM = p lasma membrane r = r i bosome S = septum t = t u b u l a r body V = v a c u o l e v = v e s i c l e W = wall ACKNOWLEDGEMENT The a u t h o r w i s h e s t o thank P r o f e s s o r C l a y t o n P e r s o n , under whose s u p e r v i s i o n t h i s s t u d y was c a r r i e d o u t , f o r h i s encouragement o f independent thought i n r e s e a r c h and h i s v a l u a b l e s u g g e s t i o n s i n the p r e p a r a t i o n o f t h i s m a n u s c r i p t . A p p r e c i a t i o n i s a l s o g i v e n t o Dr. Thana B i s a l p u t r a and Dr. K a t h l e e n C o l e , c o - s u p e r v i s o r s , f o r t h e i r a d v i c e and c r i t i c i s m s d u r i n g the i nves t i ga t i on. The v a l u a b l e t e c h n i c a l a s s i s t a n c e o f Mr. L.L. V e t o , i n m i c r o s c o p y and p h o t o g r a p h y i s g r a t e f u l l y acknowledged. Thanks t o Mr. V. Bourne f o r p h o t o g r a p h i c a s s i s t a n c e . Thanks a r e a l s o e x p r e s s e d t o o t h e r members o f the Department o f Botany who have a s s i s t e d i n the g r a d u a t e s t u d i e s programme o f the a u t h o r a t the 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 . G r a t i t u d e i s e x p r e s s e d t o M a r g a r e t Shand f o r a s s i s t a n c e w i t h c u l t u r e t e c h n i q u e s . To R i t a Rosbergen, thank you f o r t y p i n g the t h e s i s . L a s t but by f a r not l e a s t , many thanks a r e due t o the a u t h o r ' s c o l l e a g u e s f o r t h e i r c o n s t a n t encouragement and h e l p f u l d i s c u s s i o n s , and w i t h o u t whom t h i s i n v e s t i g a t i o n might not have been c o m p l e t e d . 1 INTRODUCTION Hawker (1965) p o i n t e d o ut t h a t f u n g i a r e d i f f i c u l t t o s t u d y w i t h t he l i g h t m i c r o s c o p e because t h e i r c h i t i n o u s w a l l s tend t o be impermeable t o the f i x i n g and s t a i n i n g s o l u t i o n s t h a t a r e n o r m a l l y u s e f u l i n c y t o l o g i c a l s t u d i e s w i t h o t h e r m a t e r i a l . These d i f f i c u l t i e s t o g e t h e r w i t h the s m a l l s i z e o f the f u n g a l o r g a n e l l e s have made i t a l m o s t i m p o s s i b l e - t o r e s o l v e t he i n t e r n a l s t r u c t u r e o f f u n g a l c e l l s a t the l i g h t m i c r o s c o p e l e v e l . With the advent o f the e l e c t r o n m i c r o s c o p e i t was hoped t h a t f u r t h e r e l u c i d a t i o n o f the i n t e r n a l s t r u c t u r e o f f u n g i would be p o s s i b l e . A g a i n , d i f f i c u l t i e s were e n c o u n t e r e d i n o b t a i n i n g s a t i s f a c t o r y f i x a t i o n . The use o f osmium t e t r o x i d e o f t e n gave poor r e s u l t s (Tsuda, 1956; Werner et_ aj_. , 1964b). However, G i r b a r d t (1958, 1961) o b t a i n e d q u i t e r e a s o n a b l e f i x a t i o n w i t h osmium t e t r o x i d e on P o l y s t i c t u s v e r s i c o l o r , a Homobasidiomycete, and B l o n d e l and T u r i a n ( i960) r e p o r t e d s i m i l a r s u c c e s s w i t h osmium t e t r o x i d e on a Phycomycete, A l l o m y c e s macrogynus. Good r e s u l t s were more o f t e n o b t a i n e d w i t h L u f t ' s (1956) p o t a s s i u m permanganate method (Hashimoto et_ aj_. , 1959; Moore, 1962; Werner et_ aj_. , 196na; MacLean, 1964), o r m o d i f i c a t i o n s o f t h i s method, u s i n g p o t a s s i u m permanganate f o l l o w e d by osmium t e t r o x i d e ( T h y a g a r a j a n e t a 1., 1961; Moore and M c A l e a r , 1961; Moore, 1962). Permanganate f i x a t i o n g i v e s e x c e l l e n t p r e s e r v a t i o n o f membranous s t r u c t u r e s but d e t a i l s o f the n u c l e u s a r e o b s c u r e , and ribosomes and o t h e r s t r u c t u r e s a r e not p r e s e r v e d . Weiss (1965) s t u d y i n g N e u rospora c r a s s a t r i e d a number o f f i x a t i o n p r o c e d u r e s i n c l u d i n g p o t a s s i u m permanganate, osmium t e t r o x i d e , g l u t a r a l d e h y d e and f o r m a l i n . G l u t a r a l d e h y d e and f o r m a l i n f i x e d m a t e r i a l was p o s t f i x e d i n osmium t e t r o x i d e . She r e p o r t e d t h a t i n c r e a s e d d e t a i l o f s t r u c t u r e was v i s i b l e i n m a t e r i a l f i x e d by the l a t t e r method. From the l i t e r a t u r e r e v i e w e d , i t seems o b v i o u s t h a t one f i x a t i o n p r o c e d u r e can g i v e q u i t e d i f f e r e n t r e s u l t s 2 on d i f f e r e n t f u n g i . In f a c t , a p r o c e d u r e t h a t i s s u i t a b l e f o r one s t a g e i n the l i f e c y c l e o f a fungus may be q u i t e u n s a t i s f a c t o r y f o r a n o t h e r . I t seems.that an adequate f i x a t i o n p r o c e d u r e f o r a p a r t i c u l a r fungus can o n l y be o b t a i n e d by e x p e r i m e n t i n g w i t h a l l the c o m b i n a t i o n s and v a r i a b l e s o f the a v a i l a b l e f i x a t i v e s and b u f f e r s u n t i l s u i t a b l e r e s u l t s a r e a c h i e v e d . Among the f u n g i f i r s t s t u d i e d w i t h the e l e c t r o n m i c r o s c o p e were y e a s t s , c e r t a i n o t h e r Ascomycetes'and some Phycomycetes (Bartholomew and M i t t w e r , 1 9 5 4 ; M e i e r and Webster, 1 9 5 4 ; Bartholomew and L e v i n , 1 9 5 5 ; Tsuda, 1 9 5 5 , 1 9 5 6 ; T u r i a n and K e l 1 e n b e r g e r , 1 9 5 6 ; Agar and D o u g l a s , 1 9 5 5 , 1 9 5 7 ; Kawakami and N e h i r a , 1 9 5 8 , 1 9 5 9 ; Y o t s u y a n a g i , 1 9 5 9 ; S h a t k i n and Tatum, 1 9 5 9 ; Edwards e t aj_. , 1 9 5 9 ; B a k e r s p i g e l , 1 9 5 9 ; Hash imoto et_ aj_. , 1 9 5 8 , 1 9 5 9 , 1 9 6 0 ; C o n t i and N a y l o r , 1 9 5 9 , 1 9 6 0 ; B l o n d e l and T u r i a n , 1 9 6 0 ; Aronson and P r e s t o n , 1 9 6 0 ; H i r a n o and L i n d e g r e n , 1961 ; T h y a g a r a j a n et_ aj_. , 1961 , 1 9 6 3 ) . With i m p r o v i n g t e c h n i q u e s a c o n s i d e r a b l e amount o f work has been done on. r e p r e s e n t a t i v e s o f a l l t he major groups of f u n g i : Phycomycetes: ( B l o n d e l and T u r i a n , 1 9 6 0 ; Moore and M c A l e a r , 1 9 6 1 , 1 9 6 3 ; Hawker and A b b o t t , 1 9 6 3 a , b; Peyton and Bowen, 1 9 6 3 ; B e r l i n and Bowen, 1 9 6 4 a ; Moore, 196*+; Werner aj_- , 1 9 6 4 a ; E h r l i c h and E h r l i c h , 1 9 6 6 ; Hawker and Gooday, 1 9 6 6; W i l l i a m s and Yukawa, 1 9 6 7 , e t c . ) ; A s comycetes: (Moore and M c A l e a r , 1 9 6 1 , 1 9 6 2 a , 1 9 6 3 ; L i n n a n e et_ aj_. , 1 9 6 2 ; H i r a n o and L i n d e g r e n , 1 9 6 3; C o n t i et_ aj_. , 1 9 6 3 ; E h r l i c h and E h r l i c h , 1 9 6 3 a ; MacLean, 1 9 6 4 ; Lowry and Sussman, 1 9 6 4 ; B e r l i n and Bowen, 1 9 6 4 b ; T s u k a h a r a and Yamada, 1 9 6 5 ; B r a c k e r , I 9 6 8 ; A k a i e_t aj_. , 1 9 6 8; C a r r o l l , 1 9 6 9 ; Kunoh and A k a i , 1 9 6 9, e t c . ) ; B a s i d i o m y c e t e s : ( B a k e r s p i g e l , 1959; Moore and M c A l e a r , 1 9 6 1 , 1 9 6 2 , 1 9 6 3 ; B r a c k e r and B u t l e r , 1 9 6 3 ; E h r l i c h and E h r l i c h , 1 9 6 3 b ; Moore, 1 9 6 3 a , b; V o e l z and N i e d e r p r u e m , 1 9 6 4 ; Boyer and I s s a c , 1 9 6 4 ; W i l l i a m s and Ledingham, 1 9 6 4 ; W e l l s , 1 9 6 4 a , b; 1 9 6 5 ; Lu, 1 9 6 6 a , b; Hyde and Walkinshaw, 1 9 6 6; Chet e t a 1 . , 1 9 6 9 ; K o l t i n and F l e x e r , 1 9 6 9 , e t c . ) . A few s t u d i e s have been co n c e r n e d w i t h the Fungi I m p e r f e c t i (Werner e t a_l_., 1 9 6 4 b ; B a l l e s t a and V i l l a n u e v a , 1 9 6 6 ) . Of t h e B a s i d i o m y c e t e s most o f the work has been done on the Homobasidio-3 mycetes, and on the U r e d i n a l e s ( r u s t s ) and T r e m e l l a l e s of the H e t e r o b a s i d i o -mycetes. Only one a c c o u n t o f u l t r a s t r u c t u r a l s t u d i e s on a member o f the U s t i 1 a g i n a 1 e s has been r e p o r t e d , t h a t o f A n t h r a c o i d e a (Kukkonen and V a i s s a l o , 1 9 6 ^ )• The m i c r o g r a p h s from t h i s s t u d y i n d i c a t e d v e r y poor f i x a t i o n . To the b e s t of the a u t h o r ' s knowledge no o t h e r work has been done on the f i n e s t r u c t u r e o f smut f u n g i . Perhaps a t t e m p t s have been made, but then abandoned because of the d i f f i c u l t i e s e n c o u n t e r e d w i t h f i x a t i o n . E x c e p t f o r p ' l a s t i d s , most o r g a n e l l e s found i n the c e l l s o f h i g h e r p l a n t s have a l s o been found i n f u n g a l c e l l s : n u c l e u s (Moore, 1 9 6 2 , 1 9 6 4 ; Hawker and A b b o t t , 1 9 6 3 a ; C o n t i e_t_ aj_. , 1 9 6 3 ; E h r l i c h and Eh r 1 i ch , 1 9 6 3; V o e l z and Niedepruem, 1 9 6 4 ) ; n u c l e o l i (Edwards et_ a_h , 1 9 5 9 ; G i r b a r d t , 1 9 6 1 ; C o n t i et_ aj_. , 1 9 6 3 ; S h a t l a <et_ a_l_. , 1 9 6 6 ; Van Dyke and Hooker, 1 9 6 9 ) ; m i c r o t u b u 1 e s ( S h a t l a et^ aj_. , 1 9 6 6; W i l l i a m s and Yukawa, 1 9 6 7; G i r b a r d t , 1 9 6 9 ) ; u b i q u i t o u s m i t o c h o n d r i a (Hash i moto et_ aj_. , 1 9 5 9 ; T h y a g a r a j a n , 1 9 6 1 ; Moore and M c A l e a r , 1 9 6 3 ; H i r a n o and L i n d e g r e n , 1 9 6 3 ; Werner e t a l . , 1 9 6 4 a , b ) . V a r i a t i o n s of c i s t e r n a l o r v e s i c u l a r s t r u c t u r e s have been r e c o g n i z e d as e n d o p l a s m i c r e t i c u l u m . Most e n d o p l a s m i c r e t i c u l u m i n f u n g i i s smooth (Moore, 1 9 6 2 ; Werner e t a l . , 1 9 6 4 a , b; E h r l i c h and E h r l i c h , 1 9 6 6 ) , though some rough e n d o p l a s m i c r e t i c u l u m has been r e p o r t e d (Peyton and Bowen, 1 9 6 3 ; B e r l i n and Bowen, 1 9 6 4 ) . C o n t i n u o u s c o n n e c t i o n s between the e n d o p l a s m i c r e t i c u l u m and the n u c l e a r e n v e l o p e have been r e p o r t e d f o r q u i t e a number of f u n g i (Edwards e t a 1 . , 1 9 5 9 ; Edwards and Edwards, 1 9 6 0 ; Moore, 1 9 6 2 ; T h y a g a r a j a n e t a 1. , 1 9 6 3 ; Hawker and A b b o t t , 1 9 6 3 b ) . Some f u n g i c o n t a i n s t r u c t u r e s r e s e m b l i n g the G o l g i a p p a r a t u s or d i c t y o s o m e s ( B l o n d e l and T u r i a n , 1 9 6 0 ; Moore and M c A l e a r , 1 9 6 2 b ; Moore, 1 9 6 3 a , b; Hawker and A b b o t t , 1 9 6 3 b ; Lu, 1 9 6 6 a ; Shat 1 a et_ aj_. , I966; E h r l i c h and E h r l i c h , 1 9 6 6 ) . W i t h improvements i n f i x a t i o n , ribosomes a r e now w i d e l y r e p o r t e d ( B l o n d e l and T u r i a n , I96O; W e i s s , 1 9 6 5 ; Hyde and Walkinshaw, 1 9 6 6 ; Chet et^ a_l_. , 1969) - Lomasomes, named by Moore and M c A l e a r (1961) were a t f i r s t t h o ught t o be r e s t r i c t e d t o f u n g i ( P e y t o n and Bowen, 1 9 6 3 ; B e r l i n and Bowen, 1 9 6 4 a ) but have now been o b s e r v e d i n h i g h e r p l a n t s (Manocha and Shaw, 1 9 6 4 ) . For a more d e t a i l e d a c c o u n t see Marchant and Robards ( 1 9 6 8 ) . The most common s t o r a g e p r o d u c t s i n f u n g i a r e l i p i d s ( W e l l s , 1 9 6 4 b ; W e i s s , 1 9 6 5 ; W i l l i a m s and Yukawa, 1 9 67 ) and g l y c o g e n (Werner e_t_ aj_. , 1 9 6 4 a , b; W e l l s , 1 9 6 5 ; A k a i e_t_ a_l_. , 1 9 6 8 ) . S e p t a , c o m p l e t e or p o r o u s , a r e c h a r a c t e r i s t i c of many f u n g i (Moore and M c A l e a r , 1 9 6 2 a ; B r a c k e r and B u t l e r , 1 9 6 3 ; W e l l s , 1 9 6 4 a , 1 9 6 5 ; B a l l e s t a and V i l l a n u e v a , 1 9 6 6 ; B r a c k e r , 1 9 6 8 ) . V a c u o l e s o f v a r i o u s s i z e s and numbers o c c u r i n a l l f u n g i s t u d i e d , but the s i z e and number depends on the s t a g e i n the l i f e c y c l e o f the fungus and the age. T h i s i s by no means an e x h a u s t i v e s u r v e y of u 1 t r a s t r u c t u r a 1 s t u d i e s done on f u n g i but more d e t a i l e d and e x t e n s i v e i n f o r m a t i o n can be found i n the r e v i e w a r t i c l e s by C a r b o n e l l and P o l l a k ( 1 9 5 9 - 1 9 6 3 ) , Hawker ( 1 9 6 5 ) and B r a c k e r ( 1 9 6 7 ) -The e a r l y s t u d i e s o f f u n g a l s t r u c t u r e were c a r r i e d , out u s i n g l i g h t m i c r o s c o p y . These i n c l u d e t h e v e r y a c c u r a t e and d e t a i l e d s t u d y o f v a r i o u s members o f t h e U s t i l a g i n a l e s w h i c h was r e p o r t e d by Wang ( 1 9 3 4 ) . She d e s c r i b e d young m y c e l i a l f i l a m e n t s of Ust i 1 ago horde i ( P e r s . ) L a g e r h . as a p p e a r i n g long and t h i n , and c o n t a i n i n g one o r more n u c l e i . F i l a m e n t s were seen t o be c o n t r a c t e d i n c e r t a i n p l a c e s , and s w e l l e d i n o t h e r s where the c y t o p l a s m had a c c u m u l a t e d . P a r t i t i o n s formed t o s e p a r a t e the c o n t r a c t e d p a r t s from the s w o l l e n p a r t s . Through use o f s p e c i f i c v i t a l s t a i n s Wang a l s o s t u d i e d the growth and the-movements of m i t o c h o n d r i a and v a c u o l e s . S i n c e no o t h e r d e t a i l e d c y t o l o g i c a l work has s i n c e been c a r r i e d o u t , the purpose o f t h i s i n v e s t i g a t i o n was t o s t u d y a m y c e l i a l c o l o n y o f Ust i 1 ago horde i t o f u r t h e r e l u c i d a t e the i n t e r n a l s t r u c t u r e o f the hyphae, t o v e r i f y and ex t e n d the r e s u l t s o b t a i n e d e a r l i e r by Wang, and t o compare the u l t r a s t r u c t u r e of Ust i 1 ago wi t h t h a t o f o t h e r f u n g i , s p e c i f i c a l l y w i t h r e g a r d t o the number of n u c l e i per c e l l , and the n a t u r e of the p a r t i t i o n s o r s e p t a . 5 MATERIALS AND METHODS MATERIAL: The m y c e l i a l c o l o n y o f Us t i1 ago horde i ( P e r s . ) Lagerh. used i n t h i s s t u d y was a mutant i s o l a t e d by Dr. Jean Mayo from a c o m p l e m e n t a t i o n t e s t i n whic h the p a r e n t s were a r g i n i n e homozyg'otes w i t h the f o l l o w i n g a u x o t r o p h i c m a r k e r s : 1.1 Met Pan Arg A x 1.4 M e t + P a n + Arg a. From the c o m p l e m e n t a t i o n t e s t p l a t e the m y c e l i a l c o l o n y was s u b c u l t u r e d i n t o c o m p l e t e and mini m a l shake c u l t u r e s and then o n t o c o m p l e t e and mini m a l agar p l a t e s . For s t o r a g e the c u l t u r e s were p l a c e d i n s i l i c a g e l . The b a s i s f o r the c o m p l e m e n t a t i o n t e s t i s t h a t n e i t h e r o f the monokaryons, owing t o t h e i r s p e c i f i c n u t r i t i o n a l r e q u i r e m e n t s , can grow on mini m a l medium ( D i n o o r and P e r s o n , 1969)- T h e r e f o r e , s i n c e t he p r o d u c t o f t h e c o m p l e m e n t a t i o n t e s t was a b l e t o grow on mini m a l i t i s assumed t o be a d i k a r y o n . The c o l o n i e s p r e p a r e d f o r e l e c t r o n m i c r o s c o p y were t a k e n from a mini m a l s i l i c a g e l c u l t u r e and s u b c u l t u r e d on c o m p l e t e agar p l a t e s , m a i n t a i n e d i n an i n c u b a t o r a t 22° C f o r 5 days. The r e c i p e f o r the c u l t u r e media can be found i n App e n d i x A. A f t e r 5 days i n d i v i d u a l c o l o n i e s o f dense growth were v i s i b l e on the p l a t e s (Appendix B ) . MATING TEST: The m y c e l i a l c o l o n y was c r o s s e d w i t h t he two m a t i n g t y p e s E^ and l ^ + on c o m p l e t e a g a r p l a t e s . A f t e r 48 hours o f i n c u b a t i o n a t 22° C the p l a t e s were r e a d . T h i s t e s t was c a r r i e d o u t t h r e e t i m e s . LIGHT MICROSCOPY: T h i c k s e c t i o n s (i y) o f epoxy embedded m a t e r i a l were s t a i n e d w i t h T o l u i d i n e B l u e (Appendix C) and examined on a Z e i s s P h o t o m i c r o s c o p e . A l s o squash p r e p a r a t i o n s o f the m y c e l i a l c o l o n y from shake and p l a t e c u l t u r e s were s t u d i e d w i t h t he l i g h t m i c r o s c o p e . U n s t a i n e d p r e p a r a t i o n s and t h o s e 6 stained with prop ion ic - i ron haematoxy1in (Henderson and Lu, 1968, Appendix D) were photographed with a Zeiss photomicroscope using phase contrast or ordinary transmission. PREPARATION FOR ELECTRON MICROSCOPY: Colonies were f ixed for electron microscopy at room temperature using the fol lowing procedures: (1) 1.5% unbuffered potassium permanganate at pH ~] .h for 20 minutes (Luft , 1956) or (2) a mixture of 8% glutaraldehyde and 2% osmium tet rox ide , equal volumes of each in .01M cacodylate buffer pH 6.8 and 7.2 for 1 hour (T. B i s a l -putra, personal communication) or (3) 2.5? glutaraldehyde in .01M cacodylate buffer pH 7-2 for 1 hour and, a f te r washing in buffer , postfixed in \% osmium tetroxide for 1 hour (C.E. Bracker and S.N. Grove, personal communication). Plates with colonies were flooded with f i x a t i v e , and the material minced with razor blades. The minced material was then transferred to v i a l s of fresh f i x a t i v e . Following f i x a t i o n the material was washed for 1 hour in buf fer , in the case of procedures (2) and (3), and in d i s t i l l e d water for procedure (1). Mater ial f ixed by procedure (3) was stained in 0.5? aqueous uranyl acetate (Watson, 1958) for 2 hours pr ior to dehydration. A l l material was dehydrated through a graded ser ies of ethanol-propy1ene oxide so lu t ions . Epon 812 (Luft , 1961) was used for i n f i l t r a t i o n and embedding of the mater ia l . Polymerization of the blocks was carr ied out for 12 hours at 37° C and 30 hours at 60° C. S i l ve r to grey coloured sections were cut on Sorval l Porter-Blum MT-2 ultramierotome using hand-broken glass knives. Sections were picked up on carbon-coated copper g r ids . Sections cut from material f ixed by procedures (1) and (2) were post -stained in a saturated so lut ion of uranyl acetate in 70? ethanol for 20-25 minutes, followed by lead c i t r a t e (Reynolds, 1963) for 20-30 minutes. Sections from procedure (3) material were post -stained 7 i n l e a d c i t r a t e o n l y , as above. A l l s e c t i o n s were viewed w i t h a H i t a c h i HS-75 o r H i t a c h i HU-11A m i c r o s c o p e a t 50 o r 75 KV. RESULTS I . Ma t i ng T e s t . The mutant myce 1 i a 1 s t r a i n o f U_. h o r d e i gave a p o s i t i v e m a t i n g r e a c t i o n when i t was t e s t e d i n c o m b i n a t i o n w i t h the s t a n d a r d p l u s s t r a i n ( l ^ + ) . A l t h o u g h t h i s may be taken as e v i d e n c e t h a t the m y c e l i a l s t r a i n was a h a p l o i d minus s t r a i n , the e v i d e n c e cannot be c o n c l u s i v e s i n c e the p o s s i b i l i t y t h a t the s t r a i n was an u n s t a b l e d i-ka<ryon,. which- formed buds o f o n l y one m a t i n g t y p e ( i . e . m i n u s ) , has not been e x c l u d e d (McLaren, 1 9 6 7 ) • I I . L i g h t M i c r o s c o p y . The mutant m y c e l i a l c o l o n y o f U. h o r d e i c o n s i s t s o f numerous long f i l a m e n t o u s hyphae wh i c h v a r y c o n s i d e r a b l y i n d i a m e t e r . Most hyphae a r e narrow w i t h v e r y l o n g c e l l s , w h i l e o t h e r s appear somewhat s w o l l e n ( F i g . 1) w i t h c o n s t r i c t i o n s a t some p o i n t s a l o n g the hypha ( F i g . 3, a r r o w ) . Some hyphae appear t o have v e r y few s e p t a , w h i l e o t h e r s seem to be r e g u l a r l y s e p t a t e d ( F i g . 1). The few branches t h a t a r e o b s e r v e d ( F i g . 1, arrow) a l w a y s o c c u r a t the p o i n t where a septum o r c r o s s w a l l i s p r e s e n t . Very few o r g a n e l l e s can be d i s t i n g u i s h e d . U s i n g phase c o n t r a s t some v a c u o l e s a r e o b s e r v e d , and some s m a l l e r , more dense b o d i e s w h i c h a r e p r o b a b l y m i t o c h o n d r i a . P r o p i o n i c -i r o n haematoxy1in s t a i n s n u c l e i and m i t o c h o n d r i a q u i t e p r o m i n e n t l y ( F i g s . 2, 3 ) - S i n c e no o t h e r s p e c i f i c s t a i n s were used l i t t l e e l s e w i t h i n the f u n g a l c e l l c o u l d be i d e n t i f i e d w i t h the l i g h t m i c r o s c o p e . III. E l e c t r o n M i c r o s c o p y . The f i x a t i o n method o f B r a c k e r and Grove w h i c h employs c a c o d y l a t e b u f f e r e d g l u t a r a l d e h y d e f o l l o w e d by p o s t f i x a t i o n in osmium t e t r o x i d e gave the b e s t 9 p r e s e r v a t i o n o f hyphal s t r u c t u r e . P o t a s s i u m permanganate w h i c h gave e x c e l l e n t p r e s e r v a t i o n o f membranes was used as an a l t e r n a t i v e method f o r c o m p a r a t i v e u l t r a s t r u c t u r e and t o t e s t f o r f i x a t i o n a r t i f a c t s . The m i x t u r e o f g1utara1dehyde-osmiurn t e t r o x i d e gave adequate f i x a t i o n but not as much f i n e d e t a i l as B r a c k e r and Grove's method. C e l l s o f the hyphae a r e v e r y l o n g . T h i s i s i n agreement w i t h Wang's d e s c r i p t i o n (1934). An e n t i r e c e l l , d e f i n e d as the s e c t i o n o f a h y p h a . between two c r o s s w a l l s , was never seen w i t h the e l e c t r o n m i c r o s c o p e , however the l e n g t h o f a s i n g l e c e l l d e t e r m i n e d by l i g h t m i c r o s c o p y ranged w i d e l y , from 1.1 t o 40 y. I t seems t h a t c r o s s w a l l s o r s e p t a a r e not p r e s e n t to d e l i n e a t e c e l l s , but r a t h e r t o s e a l o f f d e g e n e r a t e a r e a s . T h i s a s p e c t o f s e p t a t i o n w i l l be d i s c u s s e d f u r t h e r i n a l a t e r s e c t i o n . A. A p i c a l r e g i o n s of hyphae. The w i d t h o f a p i c a l r e g i o n s o f hyphae ranges from 2.0 t o 5-0 y. A f a i r l y narrow w a l l , about 61 my t h i c k s u r r o u n d s the hypha (W, F i g s , 4 and 5). I t i s s t r a i g h t and q u i t e - smooth- on the o u t e r s u r f a c e . O c c a s i o n a l l y a t h i c k e n i n g o r s w e l l i n g i s seen a l o n g t h e i n n e r s u r f a c e o f the w a l l (X, F i g . 5). An a p p a r e n t i n c l u s i o n o f more d e n s e l y s t a i n e d m a t e r i a l o c c u r s i n the w a l l a t t h i s p o i n t . At the m a g n i f i c a t i o n s used no s u b s t r u c t u r e was v i s i b l e i n the w a l l , nor d i d t h e w a l l appear t o c o n s i s t of l a y e r s . S e n t a n d r e u and V i l l a n u e v a (1965) r e p o r t e d a three-1ayered w a l l f o r Cand i da ut i1i s , the i n n e r l a y e r b e i n g l a m i n a t e d . These l a m i n a t i o n s were thought t o be a s s o c i a t e d w i t h the p r e s e n c e o f c h i t i n , however such l a m i n a t i o n s were not seen i n t h e w a l l o f U_. h o r d e i . Sometimes the w a l l i s v e r y w e a k l y s t a i n e d and a l m o s t e l e c t r o n t r a n s p a r e n t ( F i g s , 4 and 7) but g e n e r a l l y i t i s o f medium e l e c t r o n d e n s i t y and f i n e l y g r a n u l a r ( F i g s . 5 and 8). A d i s t i n c t plasma membrane o f t h e s i n g l e " u n i t membrane" t y p e i s p r e s e n t ( P M , F i g s . 4, 5 and 8). The t h i c k n e s s v a r i e s from 110A t o 120A. T h i s measurement i s s i m i l a r t o t h a t r e p o r t e d f o r F u l i g o s e p t i c a (Corfman, 1966). The plasma membrane o f U_. horde i i s e l e c t r o n dense, h i g h l y c o n v o l u t e d and has numerous i n v a g i n a t i o n s , (Y, F i g s , h and 5) l i k e the plasma membrane of N e u r o s p o r a c r a s s a ( S h a t k i n and Tatum, 1959), but i t i s a l m o s t t w i c e as t h i c k . Edwards et_ aj_. (1959) a l s o r e p o r t e d an e l e c t r o n dense, r u g o s e , v e s i c u l a t e d plasma membrane f o r H i s t o p 1asma c e l l s i n c u l t u r e . O c c a s i o n a l l y the plasma membrane of U_. h o r d e i runs a l o n g s m o o t h l y , c l o s e l y a d h e r i n g t o the w a l l ( F i g s . 7 and 8). The c y t o p l a s m i s d e n s e l y g r a n u l a r and c o n t a i n s numerous ribosomes ( r , F i g s . 5 and 9). L i p i d s (Hawker, 1965) o c c u r i n the a p i c a l r e g i o n s of the hyphae ( L , F i g s . 6, 8 and 9)- L i p i d d r o p l e t s measured from 0.3 t o 0.7 u i n d i a m e t e r . L a r g e r l i p i d d r o p l e t s a p p r o a c h i n g 1 y i n d i a m e t e r were o c c a s i o n a l l y seen i n permanganate f i x e d m a t e r i a l . I n f r e q u e n t l y , a r e a s o f c y t o p l a s m w i t h i n the a p i c a l r e g i o n o f a hypha appeared f l o c c u l e n t ( F, Fig.7). The n a t u r e of t h e e l e c t r o n t r a n s p a r e n t a r e a s r e s p o n s i b l e f o r the f l o c c u l e n t a p p e a r a n c e i s unknown. C l u s t e r s o f dense g r a n u l e s , 300 A i n d i a m e t e r were o b s e r v e d i n the c y t o p l a s m (G, F i g . k); t h e s i z e and a p p e a r a n c e o f t h e s e g r a n u l e s s u g g e s t t h a t they might be g l y c o g e n (Werner e t a 1., 1966; McKeen e t a 1 . , 1967 ; Ehr 1 i ch et_ aj_. , 1 968) . S i nee no' h i stochemi ca 1 s t u d y was c a r r i e d o u t , the g r a n u l e s cannot be c a l l e d g l y c o g e n w i t h c e r t a i n t y . M i c r o -t u b u l e s ( S h a t l a e_t_ aj_. , 1966) were n o t i c e d q u i t e f r e q u e n t l y (MT, F i g . k) , u s u a l l y r u n n i n g p a r a l l e l w i t h a n u c l e a r membrane o r c l o s e t o the plasma membrane. No t y p i c a l lomasomes (Moore and M c A l e a r , 1961) were seen. A few u n i d e n t i f i e d b o d i e s ( i , F i g s . 5 and 8) w h i c h may be m i c r o b o d i e s were o b s e r v e d o c c a s i o n a l l y . No hyphal b r a n c h i n g o c c u r s i n the a p i c a l r e g i o n s . The m y c e l i a l c o l o n y o f U. h o r d e i used i n t h i s s t u d y i s d i k a r y o t i c as d e f i n e d e a r l i e r i n M a t e r i a l s and Methods, t h e r e f o r e one would e x p e c t t o f i n d two n u c l e i i n each n o n - d i v i d i n g hyphal c e l l . However, t h i s s i t u a t i o n was o b s e r v e d o n l y o c c a s i o n a l l y i n the a p i c a l r e g i o n s (N, F i g . 1 2 ) , and some-11 times f u r t h e r back from the a p i c a l t i p (N, F i g . 1 3 ) - The n u c l e i a r e v e r y l o n g and somewhat d i f f u s e i n app e a r a n c e . N u c l e i o f m a t e r i a l f i x e d w i t h g 1utara 1dehyde-osmiurn t e t r o x i d e a r e u n i f o r m l y g r a n u l a r , and as e l e c t r o n dense as t h e s u r r o u n d i n g c y t o p l a s m (N, F i g s , k and 9 ) . There i s v e r y l i t t l e c o n t r a s t , i f any, between the d e n s i t y o f the n u c l e o p l a s m and the c y t o p l a s m . O c c a s i o n a l l y the n u c l e i appear more e l e c t r o n opaque than g r a n u l a r . In permanganate f i x e d m a t e r i a l the n u c l e o p l a s m i s g e n e r a l l y f i n e l y g r a n u l a r and as dense as the c y t o p l a s m (N, F i g s . 6 , 8 and 11) but o c c a s i o n a l l y more dense (N, F i g . 1 2 ) . S c a t t e r e d t h r o u g h o u t the n u c l e o p l a s m a r e e l e c t r o n t r a n s p a r e n t a r e a s w h i c h r e p r e s e n t c h r o m a t i n t h a t has not been p r e s e r v e d by permanganate. The long s i n u o u s a ppearance o f the n u c l e i p r o b a b l y c o r r e l a t e s w i t h the c y t o p l a s m i c s t r e a m i n g i n the f u n g u s , w h i c h i n t u r n i s a s s o c i a t e d w i t h t h e a p i c a l form o f growth. N u c l e a r l e n g t h i s t h e r e f o r e h i g h l y v a r i a b l e ; n u c l e a r w i d t h i s , however, more c o n s t a n t , u s u a l l y 1 t o 2 y. I t i s al w a y s g r e a t e r than h a l f the w i d t h o f the hypha. A f a i r l y l a r g e n u c l e o l u s i s p r e s e n t i n t he n u c l e u s (NU, F i g . 4 ) . I t seems t o be v e r y g r a n u l a r around i t s p e r i p h e r y , and more f i b r o u s toward i t s c e n t e r . These r a t h e r p o o r l y d e f i n e d a r e a s may r e p r e s e n t t h e pars g r a n u l o s a and pars f i b r o s a , r e s p e c t i v e l y . The n u c l e o l u s i n f i g u r e h measures a p p r o x i m a t e l y 1 y. Sometimes the p r e s e n c e o f a n u c l e o l u s i s f a i n t l y s u g g e s t e d . The n u c l e o l u s does not have a membrane. An uneven o r u n d u l a t i n g d o u b l e membrane s u r r o u n d s the n u c l e u s (NE, F i g s . 4, 9 and 11). As w i t h the d i m e n s i o n s o f the o t h e r o r g a n e l l e s , t h e t h i c k n e s s o f the n u c l e a r e n v e l o p e v a r i e s c o n s i d e r a b l y . The t o t a l t h i c k n e s s v a r i e s from 225-300 A, w i t h each membrane about 85 t o 110 A t h i c k . A p e r i n u c l e a r space between the two membranes measures from 55 t o 85 A. The n u c l e a r e n v e l o p e i s al w a y s r a t h e r i n d i s t i n c t , p o s s i b l y due t o i n a d e q u a t e f i x a t i o n o r s t a i n i n g , so t h a t v e r y few n u c l e a r p o r e s a r e seen. N u c l e a r p o r e s , i n f a c t , were o b s e r v e d w i t h c e r t a i n t y o n l y i n permanganate f i x e d m a t e r i a l (NP, F i g s . 6 and 11). These pores a r e o s i m p l e , and 500 t o 800 A i n d i a m e t e r . No f u r t h e r s t r u c t u r a l d e t a i l s o f the pores were v i s i b l e . N e i t h e r n u c l e a r b l e b b i n g nor i n t e r c o n n e c t i o n s between the n u c l e a r e n v e l o p e and c e r t a i n o r g a n e l l e s were seen i n the c e l l s o f the mutant m y c e l i a l c o l o n y o f U. h o r d e i . P o s s i b l y some n u c l e a r b l e b b i n g c o u l d o c c u r from the long p r o j e c t i o n s o f the n u c l e u s (P, F i g s . 11 and 12) t h a t a r e sometimes seen. The m i t o c h o n d r i o n i s the most prominent and the most numerous o r g a n e l l e i n a p i c a l r e g i o n s . A t y p i c a l m i t o c h o n d r i o n i s v e r y e l o n g a t e (M, F i g . 10). The w i d t h ranges from 0.2 t o 0.6 u i n m a t e r i a l f i x e d w i t h g l u t a r a l d e h y d e -osmium t e t r o x i d e , and t o the somewhat l a r g e r d i m e n s i o n s of 0.75 t o 0.95 y in permanganate f i x e d m a t e r i a l . Some o f the m i t o c h o n d r i a i n Saccharomyces d e s c r i b e d by H i r a n o and L i n d e g r e n (19 61) a r e s i m i l a r t o t h o s e i n U. horde i . G e n e r a l l y the m i t o c h o n d r i a a r e o r i e n t e d l o n g i t u d i n a l l y w i t h r e s p e c t t o the hyphal c e l l . Each m i t o c h o n d r i o n i s s u r r o u n d e d by a c h a r a c t e r i s t i c d o u b l e o membrane. The o u t e r membrane, 60 t o 140 A i n w i d t h , i s c o n t i n u o u s around the m i t o c h o n d r i o n . The i n n e r membrane 50 t o 120 A w i d e , i n v a g i n a t e s t o form the p l a t e - l i k e c r i s t a e . Osumi (1965) r e p o r t e d m i t o c h o n d r i a l membrane d i m e n s i o n s o v e r a s i m i l a r range f o r j>accha romyces. The p l a t e - l i k e c r i s t a e o f U_. horde i l i e p a r a l l e l t o the long a x i s o f the m i t o c h o n d r i o n ; t h i s i s an unus u a l s i t u a t i o n f o r the f u n g i , a l t h o u g h s i m i l a r d e s c r i p t i o n s a r e g i v e n f o r the c r i s t a e o f P u c c i n i a s o r g h i (Van Dyke and Hooker, 1969) and of Neurospora c r a s s a ( S h a t k i n and Ta turn, 1959) • C r i s t a e o f U_. horde i measu re 18 t o 25 my i n w i d t h . The g r a n u l a r m a t r i x i s u s u a l l y s l i g h t l y more e l e c t r o n dense than the o c y t o p l a s m . Very s m a l l g r a n u l e s , 100 t o 175 A i n d i a m e t e r a r e s c a t t e r e d t h r o u g h o u t the m a t r i x ( g , F i g . 10). Some v a c u o l e s a r e p r e s e n t i n the a p i c a l r e g i o n s . These range i n s i z e from 0.25 t o 2.0 y, most of them i n t h e lower l i m i t o f the range, and can be round o r o v o i d i n shape (V, F i g s , h, 5 and 6). G e n e r a l l y , the v a c u o l e s a r e e l e c t r o n t r a n s p a r e n t and bound by a s i n g l e u n i t membrane about 110 A t h i c k . The v a c u o l e s may appear empty ( F i g . 4) o r c o n t a i n s u b s t a n c e s o f medium e l e c t r o n d e n s i t y ( F i g . 5)- Uptake o f m a t e r i a l i n t o the v a c u o l e s seems t o be by i n v a g i n a t i o n o f the t o n o p l a s t ( F i g . 17, a r r o w ) . . The term " v e s i c l e s " i s used t o d e s c r i b e s m a l l e l e c t r o n t r a n s p a r e n t b o d i e s s i m i l a r t o v a c u o l e s but c o n s i d e r a b l y s m a l l e r i n s i z e . These v e s i c l e s ( v, F i g . 4) a r e s u r r o u n d e d by s i n g l e u n i t membranes; they a r e empty i n a p p e a r a n c e , and they range i n s i z e from .08 t o .15 p. T h e i r f u n c t i o n i s unknown but i t i s thought t h a t they a r e p r o b a b l y p r e c u r s o r s o f l a r g e r v a c u o l e s . E n d o p l a s m i c r e t i c u l u m i s f a i r l y s p a r s e i n a p i c a l r e g i o n s , and u s u a l l y more d i s t i n c t i n permanganate f i x e d m a t e r i a l (ER, F i g s . 6 and 8) than i n g l u t a r a l d e h y d e - o s m i u r n t e t r o x i d e f i x e d m a t e r i a l (ER, F i g . 5). The appearance o f the e n d o p l a s m i c r e t i c u l u m i n permanganate f i x e d m a t e r i a l i s v e r y s i m i l a r t o the e n d o p l a s m i c r e t i c u l u m o f P y r e n o c h a e t a t e r r e s t r i s (Hess, 1969). I t i s o f the c i s t e r n a l t y p e c h a r a c t e r i z e d by s h o r t f l a t t e n e d s a c - l i k e s t r u c t u r e s . In the c e n t e r i s the i n t e r c i s t e r n a 1 s p a c e , 80 t o 110 A t h i c k , bound by a 0 membrane about 80 t o 100 A t h i c k . G e n e r a l l y the e n d o p l a s m i c r e t i c u l u m i s found c l o s e t o the plasma membrane ( F i g s . 5 and 6), i s smooth s u r f a c e d , and u s u a l l y i n an a r e a abundant i n r i b o s o m e s . Dictyosomes ( B r a c k e r , 1967) a r e now q u i t e commonly r e p o r t e d t o o c c u r i n the v a r i o u s groups o f f u n g i . However, no d i c t y o s o m e s nor s t r u c t u r e s r e s e m b l i n g d i c t y o s o m e s a r e seen i n U_. horde i . Kukkonen and V a i s s a l o (1964) i n a s t u d y o f the f i n e s t r u c t u r e o f the smut fungus A n t h r a c o i d e a mentioned t h e o c c u r r e n c e o f d i c t y o s o m e s i n t h a t f u n g u s . S i n c e the f i x a t i o n o f t h e i r m a t e r i a l was v e r y p o o r , t h e i r r e p o r t cannot be ta k e n as c o n c l u s i v e . Recent s t u d i e s o f P y r e n o c h a e t a t e r r e s t r i s (Hess, 1969) and o f Puce i n i a s o r g h i (Van Dyke and Hooker, 1969) r e p o r t t h a t d i c t y o s o m e s a r e not p r e s e n t i n t h e s e fung i . No s e p t a o c c u r i n the a p i c a l r e g i o n s o f the hyphae o f U. h o r d e i . B. D i f f e r e n t i a t e d r e g i o n s of hyphae. In d i f f e r e n t i a t e d a r e a s the w i d t h of the hypha i s u s u a l l y about 2.0 u, w h i c h i s somewhat l e s s than the w i d t h o f a p i c a l hyphae. The w a l l i s of the same'width as i t i s i n a p i c a l r e g i o n s , but i t i s more f r e q u e n t l y w e a k l y s t a i n e d ( F i g s . 14, 15 and 17) , so as t o be a l m o s t i n v i s i b l e i n p l a c e s . Dense i n c l u s i o n s o b s e r v e d i n the w a l l i n a p i c a l r e g i o n s a r e not seen i n more mature a r e a s . There i s no change i n the plasma membrane. It remains q u i t e c o n v o l u t e d ( P M , F i g s . 14 and 17) but o c c a s i o n a l l y i s smooth ( P M , F i g . 16). L i p i d d r o p l e t s (L, F i g s . 16, 17 and 19) a r e p r e s e n t but l e s s numerous than i n t h e a p i c a l r e g i o n s . However, a r e a s of f l o c c u l e n t c y t o p l a s m (F, F i g s . 15, 21 and 22) a r e s t i l l q u i t e n o t i c e a b l e i n mature r e g i o n s . C l u s t e r s o f g r a n u l e s , b e l i e v e d t o be g l y c o g e n , a r e s p a r s e i n t h e s e r e g i o n s . Ri>bosomes a r e s t i l l p r e s e n t i n abundance ( r , F i g . 17)- U n i d e n t i f i e d b o d i e s ( i , F i g s . 16 and 18) a r e a l s o o b s e r v e d , and as mentioned b e f o r e , t h e s e may r e p r e s e n t m i c r o b o d i e s . N u c l e i a r e o c c a s i o n a l l y seen i n mature r e g i o n s o f hyphae. These n u c l e i (N, F i g s . 13 and 15) a r e e l o n g a t e w i t h uneven n u c l e a r e n v e l o p e s , and g e n e r a l l y s i m i l a r t o t h o s e o b s e r v e d i n a p i c a l r e g i o n s . M i c r o t u b u l e s a r e o f t e n seen i n t h e c y t o p l a s m i n a s s o c i a t i o n w i t h the n u c l e u s (MT, F i g . 15). They a r e 230 t o 250 A i n d i a m e t e r and run p a r a l l e l t o t h e l o n g a x i s o f t h e n u c l e u s . W i l l i a m s and Yukawa (1967) found m i c r o t u b u l e s i n the c y t o p l a s m c l o s e t o the o u t s i d e o f the n u c l e a r e n v e l o p e i n P 1asmodiophora b r a s s i c a e , a Phycomycete. M i c r o t u b u l e s were never s e e n ' i n s i d e the n u c l e u s but were o c c a s i o n a l l y seen i n t he c y t o p l a s m near the plasma membrane (MT, F i g . 2 6 ) . Some s t r i k i n g changes a r e o b s e r v e d i n the m i t o c h o n d r i a . In some o f them the c r i s t a e form c o n c e n t r i c 1amel 1ae- 1ike s t r u c t u r e s (C, F i g . 16) o r have a somewhat d i s o r d e r l y a p p e a r a n c e . T h i s may be a f i x a t i o n a r t i f a c t , but a d i s o r d e r l y a ppearance o f the c r i s t a e i s a l s o e v i d e n t i n permanganate f i x e d m a t e r i a l (M, F i g . 18). Other m i t o c h o n d r i a have a s l i g h t b u l g e on one s i d e , w h i c h c o n t a i n s an e l e c t r o n opaque a r e a (0, F i g . 17)- S t i l l o t h e r s have a bu l g e c o n t a i n i n g a c o i l o f concentric 11ame11ae (C, F i g s . 19-20). In f i g u r e 20 the c o i l on the m i t o c h o n d r i o n i s p r o j e c t i n g i n t o a v a c u o l e . These c o i l s of c o n c e n t r i c 1ame11ae can be q u i t e l a r g e and w e l l d e v e l o p e d (C, F i g . 21). The c o n c e n t r i c l a m e l l a e complex i s a l s o seen i n a s s o c i a t i o n w i t h a membrane bounded t u b u l a r body ( t , F i g . 22) w h i c h may be l o c a t e d i n the c y t o p l a s m o r w i t h i n v a c u o l e s o f v a r i o u s s i z e s ( F i g s . 24-25). T h e r e i s a l m o s t always one or more m i t o c h o n d r i a i n c l o s e p r o x i m i t y t o t h e s e v a c u o l e s ( F i g s . 17, 19 and 23). It- appears t h a t the c o i l s of c o n c e n t r i c l a m e l l a e formed i n the m i t o -c h o n d r i a a r e e n g u l f e d by v a c u o l e s t h r o u g h the p r o c e s s o f membrane i n v a g i n a t i o n ( F i g . 20). F o l l o w i n g t h i s , the s m a l l v a c u o l e s c o n t a i n i n g c o n c e n t r i c l a m e l l a e appear t o be e n g u l f e d by l a r g e r v a c u o l e s ( F i g . 23). A f u r t h e r s t e p seems t o be the d i s s o c i a t i o n o f t h e s e c o n c e n t r i c l a m e l l a e i n some way t o form t h e t u b u l a r b o d i e s ( a r r o w s , F i g s . 22 and 25). The f i n a l s t e p appears t o be'the r e l e a s e o f the t u b u l a r b o d i e s i n t o t he space between the plasma membrane and th e c e l l w a l l (Y, a r r o w s , F i g s . 13 and 26). Small v a c u o l e s a r e more numerous i n t h e a c t i v e l y g r o w i n g r e g i o n s o f hyphae, but t h e r e i s a d e c r e a s e i n t h e i r number w i t h a c o r r e s p o n d i n g i n c r e a s e i n number o f p r o g r e s s i v e l y l a r g e r v a c u o l e s p r o c e e d i n g toward o l d e r mature a r e a s ( V , F i g s . 14-16). In t h e s e a r e a s v a c u o l e s a r e more i r r e g u l a r i n shape. The c o n t e n t s o f the v a c u o l e s v a r y from m a t e r i a l of medium e l e c t r o n d e n s i t y ( F i g s . 14 -15), t o c o i l s o f c o n c e n t r i c l a m e l l a e ( F i g . 23), o r t u b u l a r b o d i e s ( F i g . 25), o r a m i x t u r e o f the l a t t e r two ( F i g . 13). V a c u o l e s a r e u s u a l l y seen c l o s e t o m i t o c h o n d r i a ( F i g s . 16, 17 and 32), and i n a s s o c i a t i o n w i t h u n u s u a l "membrane-1amel1ae" s t r u c t u r e s (ML, F i g . 13). I t i s not known whether t h e s e membrane-lamellae complexes a r e d e r i v e d from v a c u o l e s c o n t a i n i n g c o i l s o f c o n c e n t r i c l a m e l l a e produced by the m i t o c h o n d r i a , o r whether they a r e from a d i f f e r e n t s o u r c e . Some o t h e r s t u d i e s have r e p o r t e d c o n c e n t r i c membrane s t r u c t u r e s , or m y e l i n f i g u r e s and t u b u l a r b o d i e s as f o l l o w s : B u c k l e y e t aJL (1966) i n B o t r y t i s c i n e r e a ; Hashimoto and Yo s h i d a (1966) i n G e o t r i c h u m candidum; S h a t l a e_t_ aj_. (1966) i n Aphanomyces  e u t e i ches ; Peat and Banbury (19 67) i n Phycomyces ; Thomas and I s s a c (1967) i n u r e d i a o f wheat stem r u s t ; Van Dyke and Hooker (1969) i n P u c c i n i a s o r g h i T here a l s o seems t o be a f u s i o n o f v a c u o l e s , o r an e n g u l f i n g of s m a l l e r v a c u o l e s by l a r g e ones ( F i g s . 17 and 23). At some s t a g e v a c u o l e s seem d e s t i n e d t o r e l e a s e t h e i r c o n t e n t s by f u s i n g w i t h the plasma membrane and e x t r u d i n g the m a t e r i a l i n t o the space between the plasma membrane and w a l l (Y, F i g s . 13 and 26). I t i s a p p a r e n t t h a t some breakdown o r d i s s o c i a t i o n o f m a t e r i a l t a k e s p l a c e w i t h i n the v a c u o l e b e f o r e the c o n t e n t s a r e r e l e a s e d T h i s s t r o n g l y s u g g e s t s the p r e s e n c e o f c o n s i d e r a b l e amounts o f enzymes w i t h the v a c u o l e s . In m a t u r i n g r e g i o n s v e s i c l e s a r e more numerous than they a r e i n a p i c a l r e g i o n s ( v , F i g s . 13, 14 and 20). W h i l e e n d o p l a s m i c r e t i c u l u m i s q u i t e s p a r s e i n a p i c a l a r e a s , i t i s more abundant i n o l d e r r e g i o n s (ER, F i g s . 14, 15, 17 and 19). O c c a s i o n a l l y some o f the e n d o p l a s m i c r e t i c u l u m o r a p a r t of i t appears t o be rough s u r f a c e d w i t h ribosomes a t t a c h e d (ER, F i g . 14). In f i g u r e 14 (arrow) the e n d o p l a s m i c r e t i c u l u m seems t o be b l e b b i n g o f f a ves i c l e . In the s t r i c t e s t sense o f t h e i r d e f i n i t i o n s , n e i t h e r d i c t y o s o m e s nor lomasomes were o b s e r v e d i n mature r e g i o n s o f hyphae. However, some l a r g e membrane bound s t r u c t u r e s r e s e m b l i n g t u b u l a r lomasomes ( B r a c k e r , 1967) were f r e q u e n t l y seen i n the c e n t r a l r e g i o n o f a hypha ( t , F i g . 24). C r o s s w a l l s o r s e p t a a r e p r e s e n t i n o l d e r r e g i o n s but seem t o o c c u r a t i r r e g u l a r i n t e r v a l s i n some hyphae (S, F i g s . 30-31). These s e p t a a r e s i m p l e and c o m p l e t e , c o n s i s t i n g o f two w a l l s s e p a r a t e d by a narrow s p a c e . The 1 7 t o t a l t h i c k n e s s o f a septum i s 70 t o 90 mu. The septum seems to be formed by i n v a g i n a t i o n s o f the w a l l around the p e r i p h e r y o f the hypha, w h i c h then grow i n toward the c e n t e r . S i n c e no i n t e r m e d i a t e s t a g e s o f septum f o r m a t i o n were o b s e r v e d t h i s can o n l y be s u g g e s t e d as a p o s s i b i l i t y . Very l i t t l e b r a n c h i n g of hyphae o c c u r s . When i t d o e s , i t i s always n e x t t o a c o m p l e t e septum w h i c h has formed t o s e a l o f f a d e g e n e r a t e p a r t o f a hypha (B, F i g . 27). T h i s p a u c i t y o f b r a n c h i n g i s c o n f i r m e d by l i g h t m i c r o s c o p e s t u d i e s o f u n s t a i n e d squash p r e p a r a t i o n s from the mutant m y c e l i a l c o l o n y o f U_. h o r d e i . C. Degenerated r e g i o n s o f hyphae. The s t a t e o f d e g e n e r a t i o n i s d e t e r m i n e d by the absence o r appearance o f v a r i o u s o r g a n e l l e s such as the m i t o c h o n d r i a , and by the degree of v a c u o l a t i o n . The plasma membrane o f d e g e n e r a t e a r e a s i s h i g h l y c o n v o l u t e d ( F i g . 28) o r v e r y d i f f i c u l t t o d i s t i n g u i s h i n some cases ( F i g s . 3-0-3 0 • l-t appears t o be a b s e n t sometimes ( F i g . 34). Ribosomes a r e s t i l l p r e s e n t but not as numerous as i n p r e c e d i n g r e g i o n s ( F i g . 35). T h e r e i s l i t t l e l i p i d found i n d e g e n e r a t e r e g i o n s (L, F i g s . 29 and 3 0 -However, t h e r e seems t o be some g l y c o g e n p r e s e n t (G, F i g s . 31 and 33). A b s e n t from t h e s e r e g i o n s a r e n u c l e i , m i c r o t u b u l e s and e n d o p l a s m i c r e t i c u l u m . M i t o c h o n d r i a a r e p r e s e n t but t h e i r c r i s t a e a r e s w o l l e n or a b s e n t , and the shape o f the m i t o c h o n d r i o n i s d i s t o r t e d ( F i g s . 29 and 35)-Degenerate c e l l s a r e c h a r a c t e r i z e d by numerous v a c u o l e s and membranous s t r u c t u r e s ( F i g s . 28 and 32), and s e p a r a t e d from f u n c t i o n a l c e l l s by s e p t a e . As m entioned p r e v i o u s l y , t h e s e p t a appear s i m p l e and c o m p l e t e (S, F i g s . 31-32). The p o s s i b i l i t y t h a t a p o r e , o r pores a r e p r e s e n t i s not e x c l u d e d . | t may be a m a t t e r o f s e c t i o n i n g i n the c o r r e c t p l a n e t o see them. An o b l i q u e o r t a n g e n t i a l s e c t i o n t h r o u g h a septum r e v e a l s some o f the sub-s t r u c t u r e o f the w a l l (S, F i g . 33). The w a l l a p p e ars t o c o n s i s t o f a p a r a l l e l a r r a y o f f i b r i l s . A.complex system o f m i c r o f i b r i l s i n an amorphous m a t r i x has been s u g g e s t e d as a s t r u c t u r a l p a t t e r n f o r f u n g a l c e l l w a l l s by Aronson and P r e s t o n (1960). O c c a s i o n a l l y an e n t i r e c e l l i n a d e g e n e r a t e r e g i o n c o n t a i n s t u b u l a r b o d i e s ( t , F i g . 3 4 ) . In f i g u r e 35 a d e g e n e r a t e hypha appears t o have b u r s t open r e l e a s i n g i t s c o n t e n t s i n t o a space between o t h e r empty or d e g e n e r a t e hyphae. S i n c e t h i s phenomenon was seen v e r y i n f r e q u e n t l y , i t i s p r o b a b l y due t o f i x a t i o n damage r a t h e r than b e i n g a n a t u r a l l y o c c u r r i n g event i n the d e g e n e r a t i o n p r o c e s s . DISCUSSION The g e n e r a l morphology o f the mutant m y c e l i a l c o l o n y o f U_. h o r d e i as d e t e r m i n e d by e l e c t r o n m i c r o s c o p y c o n f i r m s the 1ight m i c r o s c o p e r e s u l t s o f Wang (1934). Young m y c e l i a l f i l a m e n t s were found t o be t h i n w i t h v e r y long c e l l s s e p t a t e d a t i r r e g u l a r i n t e r v a l s . Each c e l l c o n t a i n e d one or two n u c l e i . The a ppearance o f the hyphae was v a r i a b l e w i t h some a r e a s c o n t r a c t e d and o t h e r p a r t s s w o l l e n where the c y t o p l a s m had a c c u m u l a t e d . Some marked changes a r e o b s e r v e d t h r o u g h o u t the hyphae o f U_. horde i , p r o c e e d i n g from the a p i c a l t i p s t o the d e g e n e r a t e d r e g i o n s . Most o b v i o u s i s the i n c r e a s e i n the number o f v a c u o l e s , v e s i c l e s and e n d o p l a s m i c r e t i c u l u m , and t h e f o r m a t i o n o f t h e " m i t o c h o n d r i a l c o n c e n t r i c l a m e l l a e " . These changes o c c u r b e h i n d the a p i c a l t i p i n the d i f f e r e n t i a t e d and d e g e n e r a t e a r e a s . However', i n c r e a s e d v a c u o l i z a t i o n accompanied by changes i n number and s t r u c t u r e o f o t h e r c y t o p l a s m i c c o n s t i t u e n t s i s c h a r a c t e r i s t i c o f the growth and d i f f e r e n t i a t i o n c y c l e o f most f u n g i ( B u t l e r , 1966), but t h e n a t u r e o f the changes i s more o r l e s s s p e c i f i c f o r each genus o r s p e c i e s o f fung i . In U_. h o r d e i the w i d t h o f hyphae i n the a p i c a l r e g i o n s was somewhat g r e a t e r than the w i d t h i n mature or d e g e n e r a t e r e g i o n s . T h i s i s p r o b a b l y due t o the tendency f o r the c y t o p l a s m t o a c c u m u l a t e towards t h e g r o w i n g t i p t h e r e b y c a u s i n g i t t o be s w o l l e n . W a l l s of U_. h o r d e i appeared t o be s i n g l e l a y e r e d , more o r l e s s homogenous s t r u c t u r e s . Hyphae o f f u n g i u s u a l l y have a s i n g l e w a l l c o n s i s t i n g o f v a r i o u s numbers o f l a y e r s o r l a m i n a t i o n s o f c h i t i n o u s m i c r o f i b r i l s ( S h a t k i n and Tatum, 1959; S e n t a n d r e u and V i l l a n u e v a , 19&5; Peat and Banbury, 1967)-T h i s i s i n c o n t r a s t w i t h the f r e q u e n t l y t r i p l e - l a y e r e d w a l l s o f v a r i o u s f u n g a l s p o r e s (Lowry.and Sussman, 1964; W i l l i a m s and Ledingham, 1964; Acha e_t_ aj_. , 1966). L a m i n a t i o n s o r f i b r i l s i n the w a l l s o f f u n g i have been shown t o r e p r e s e n t c h i t i n (Aronson and P r e s t o n , 1960; S e n t a n d r e u and V i l l a n u e v a , 1965)- A f i b r i l l a r s u b s t r u c t u r e was e v i d e n t i n the w a l l o f U. horde i i n t a n g e n t i a l s e c t i o n s . T h i s i s i n agreement w i t h t he id e a t h a t f u n g a l c e l l w a l l s resemble t h o s e o f p l a n t s i n the m i c r o f i b r i l l a r s k e l e t a l s t r u c t u r e , but a r e o f d i f f e r e n t c h e m i c a l c o m p o s i t i o n (Hawker, 1965)- I t i s g e n e r a l l y h e l d t h a t new c e l l w a l l f o r m a t i o n i n f u n g i o c c u r s i n the a p i c a l r e g i o n (Hawker, 1965; B a r t n i c k i - G a r c i a and Lippman, 1969) but t h e r e i s s t i l l much c o n t r o v e r s y as t o how the. p r o c e s s a c t u a l l y o c c u r s . Lomasomes have been p o s t u l a t e d t o p l a y a r o l e i n w a l l f o r m a t i o n ( W i l s e n a c h and K e s s e l , 1965b). Marchant et_ aj_. (1967) have f u r t h e r e l a b o r a t e d the p r o c e s s t o a s s o c i a t e two v e s i c u l a r systems w i t h w a l l s y n t h e s i s : a system o f v e s i c l e s produced by the e n d o p l a s m i c r e t i c u l u m b e l i e v e d t o be r e s p o n s i b l e f o r " p r i m a r y " w a l l s y n t h e s i s , and m u l t i v e s i c u l a r b o d i e s a l s o produced by the e n d o p l a s m i c r e t i c u l u m b e l i e v e d t o be a s s o c i a t e d w i t h " s e c o n d a r y " w a l l s y n t h e s i s and c h i t i n d e p o s i t i o n . B r a c k e r (1967) c o n c l u d e d t h a t o r g a n i z a t i o n and growth o f the f u n g a l w a l l i s under p r o t o p l a s m i c c o n t r o l , and t h a t w a l l m a t e r i a l s o r a t l e a s t w a l l p r e c u r s o r s must t r a v e r s e the plasma membrane by some form o f s e c r e t i o n . In U. horde i s w e l l i n g s a l o n g ' t h e i n n e r w a l l c o n t a i n i n g embedded m a t e r i a l were seen i n t h e a p i c a l r e g i o n o n l y (X, F i g . 5)- At one p o i n t (upper X, F i g . 5) t h e w a l l seems t o be a b s o r b i n g some dense m a t e r i a l . T h i s may i n d i c a t e a s t a g e i n w a l l s y n t h e s i s , o r on the o t h e r hand i t may be a means of g e t t i n g r i d o f unwanted m a t e r i a l . A c o n v o l u t e d plasma membrane was c h a r a c t e r i s t i c f o r most r e g i o n s o f the hyphae o f U. h o r d e i . Such u n d u l a t i n g plasma membranes have a l s o been r e p o r t e d f o r o t h e r f u n g i (Edwards e t a 1., 1959; S h a t k i n and Tatum, 1959; 21 Hess, 1969). Hawker (1965) s t a t e d t h a t i n younger hyphal c e l l s o r i n the t i p s o f c o e n o c y t i c hyphae the plasma membrane i s n o r m a l l y c l o s e l y a d p r e s s e d to the c e l l w a l l over most o f i t s s u r f a c e , whereas i n o l d e r c e l l s , i t i s o f t e n u n d u l a t i n g w i t h o c c a s i o n a l deeper i n v a g i n a t i o n s . In some a r e a s o f the hyphae o f U_. h o r d e i the plasma membrane was c l o s e l y a d p r e s s e d t o the w a l l , but not always i n the younger hyphae. The dense g r a n u l a r c y t o p l a s m i n a p i c a l a r e a s o f U_. h o r d e i c o n t a i n s most of t he o r g a n e l l e s and i n c l u s i o n s t h a t a r e a l s o found i n o t h e r f u n g i . Some of the o r g a n e l l e s , e s p e c i a l l y the m i t o c h o n d r i a and n u c l e i , have a v e r y e l o n g a t e a p p e a r a n c e . B r a c k e r and B u t l e r (1964) a t t r i b u t e d t he e l o n g a t i o n o f m i t o c h o n d r i a and n u c l e i i n R h i z o c t o n i a s o l a n i t o the dr a w i n g out e f f e c t o f r a p i d l y moving c y t o p l a s m i n the r e g i o n o f a p o r e , but s i n c e no pores were o b s e r v e d i n U_. h o r d e i the e l o n g a t i o n o f t h e s e o r g a n e l l e s may j u s t be caused by normal c y t o p l a s m i c s t r e a m i n g i n a c t i v e l y g r o w i n g hyphae. Numerous c y t o -p l a s m i c ribosomes found i n U_. h o r d e i a r e a l s o r e p o r t e d f o r v a r i o u s o t h e r f u n g i ( B l o n d e l and T u r i a n , 1960; Peyton and Bowen, 1963; W e i s s , 1965; Hyde and Walkinshaw, 1966; Chet e t a]_. , 1969). Sussman et_ aj_. (1969) r e p o r t e d some e l e c t r o n - t r a n s p a r e n t p a t c h e s i n p e r m a n g a n a t e - f i x e d c e l l s o f g e r m i n a t i n g u r e d o s p o r e s of P u c c i n i a g r a m i n i s t r i t i c i , w h i c h gave the c y t o p l a s m a "foamy" a p p e a r a n c e . No e x p l a n a t i o n was s u g g e s t e d f o r t h i s u n i q u e f e a t u r e w h i c h r e s e m b l e s somewhat the f l o c c u l e n t c y t o p l a s m i n the a p i c a l and d i f f e r -e n t i a t e d a r e a s o f IL h o r d e i . However,the f l o c c u l e n t a r e a s o f c y t o p l a s m i n U_. h o r d e i were o b s e r v e d o n l y i n m a t e r i a l f i x e d w i t h g 1 u t a ra 1 dehyde-osm i urn t e t r o x i d e . S i n c e no h i s t o c h e m i c a l s t u d y was made o f U_. h o r d e i i t can o n l y be s u g g e s t e d t h a t t he f l o c c u l e n t a r e a s w i t h i n t he hyphae r e p r e s e n t some form o f l i p i d a c c u m u l a t i o n . L i p i d seemed t o be the major s t o r a g e p r o d u c t i n U_. h o r d e i mycel i a. A c c o r d i n g t o Hawker (1965) l i p i d i s t h e commonest and most c o n s p i c u o u s i n c l u s i o n p r o d u c t i n f u n g a l c e l l s . L i p i d i n c l u s i o n s were r e p o r t e d i n t h e l i t e r a t u r e f o r q u i t e a number of f u n g i (Hawker and A b b o t t , 1 9 6 3 a ; W e l l s , 1 9 6 4 b ; W e i s s , 1965", W i l l i a m s and Yukawa, 1 9 6 7 ) . G l y c o g e n (Werner e t a 1 . , 1 9 6 4 a , b; W e l l s , 1 9 6 5 ; Werner et_ aj_. , 1 9 6 6 ; McKeen e t aj_. , 1 9 6 7 ; E h r l i c h et_ aj_. , 1 9 6 8 ; A k a i e_t aj_. , 1 9 6 8 ) i s found o n l y i n s m a l l amounts i n LL h o r d e i i f a t a l l . C l u s t e r s o f g r a n u l e s (G, F i g . 2 7 ) 3 0 0 A i n d i a m e t e r were o b s e r v e d v e r y i n f r e q u e n t l y i n m a t e r i a 1 ' f i x e d w i t h a m i x t u r e o f g 1utara 1dehyde-osmiurn t e t r o x i d e , but not i n m a t e r i a l f i x e d w i t h t h e o t h e r two p r o c e d u r e s . On the b a s i s of s i z e and a p p e a r a n c e o n l y , t h e s e g r a n u l e s may r e p r e s e n t g l y c o g e n , o r p o s s i b l y clumped r i b o s o m e s . S i n c e B r a c k e r ( 1 9 6 7 ) r e p o r t e d t h a t g l y c o g e n i s more abundant i n mature o r aged p o r t i o n s than near the growing hyphal t i p s the g r a n u l e s o b s e r v e d i n the o l d e r hyphae of U_. h o r d e i may be g l y c o g e n . Smooth e n d o p l a s m i c r e t i c u l u m i n a n i m a l s i s u s u a l l y a s s o c i a t e d w i t h g l y c o g e n s y n t h e s i s . In f u n g i w h i c h c o n t a i n c o n s i d e r a b l e amounts o f g l y c o g e n l i t t l e i s known o f the s i t e o f g l y c o g e n s y n t h e s i s . However, Hashimoto and Y o s h i d a ( 1 9 6 6 ) r e p o r t e d g l y c o g e n s y n t h e s i s i n the t u b u l a r o r c i s t e r n a l e l ements o f a u n i q u e membranous system of t u b u 1 o - v e s i c u 1 a r s t r u c t u r e s i n the I m p e r f e c t f u n g u s , G e o t r i c h u m candidum. Lomasomes (Moore and M c A l e a r , 1 9 6 1 ) , the f r e q u e n t l y r e p o r t e d " b o r d e r b o d i e s " c h a r a c t e r i s t i c o f f u n g i , were not seen i n U_. horde i . They a r e a l s o a b s e n t from E r y s i p h e g r a m i n i s h o r d e i ( B r a c k e r , 1 9 6 8; Kunoh and A k a i , 1 9 6 9 ) . A r e c e n t p h y s i o l o g i c a l and c y t o l o g i c a l s t u d y of a r t i c h o k e t u b e r s (Fowke and S e t t e r f i e l d , 1 9 6 9 ) d e a l t w i t h the n a t u r e o f m u l t i v e s i c u l a r s t r u c t u r e s a t the plasma membrane (plasmalemmasomes, lomasomes), w i t h i n the c y t o p l a s m and w i t h i n the c e n t r a l v a c u o l e s . Fowke and S e t t e r f i e l d c o n c l u d e d t h a t plasmalemmasomes a r e not i n v o l v e d i n c e l l w a l l d e p o s i t i o n , and s u g g e s t e d t h a t some m u l t i v e s i c u l a r s t r u c t u r e s may r e p r e s e n t a r t i f a c t u a l r e o r g a n i z a t i o n o f . p l a s m a lemma and t o n o p l a s t membranes d u r i n g c y t o l o g i c a l p r o c e s s i n g . S t r u c t u r e s r e s e m b l i n g " t u b u l a r " lomasomes ( B r a c k e r , 1967) were f r e q u e n t l y seen i n the c e n t r a l r e g i o n s o f the o l d e r . hyphae o f U_. h o r d e i o r near the plasma membrane, but they a r e much l a r g e r than lomasomes r e p o r t e d i n the l i t e r a t u r e f o r o t h e r f u n g i , and a r e o n l y seen i n c l o s e a s s o c i a t i o n w i t h the plasma membrane when they f u s e w i t h i t . Such s t r u c t u r e s s i m i l a r t o tho s e found i n U_. h o r d e i have been r e p o r t e d f o r P o l y s t i c t u s v e r s i c o l o r ( G i r b a r d t , 1961); P e r o n o s p o r a m a n s h u r i c a (Peyton and Bowen, 1963); B o t r y t i s c i nerea ( B u c k l e y e_t_ aJL , 1966) ; u r e d i a o f wheat stem r u s t (Thomas and I s s a c , 1967); and P u c c i n i a s o r g h i (Van Dyke and Hooker, 1969). Marchant and Robards (1968) c o n c l u d e d t h a t t he v e s i c u l a r and membranous s t r u c t u r e s a s s o c i a t e d w i t h the . p1asma1emma i n both h i g h e r and lower p l a n t s a r e not a r t i f a c t s . They have proposed new t e r m i n o l o g y f o r , and r e c l a s s i f i c a t i o n of such s t r u c t u r e s as f o l l o w s : a l l membranous o r v e s i c u l a r s t r u c t u r e s a s s o c i a t e d w i t h t he plasmalemma be c l a s s i f i e d under the g e n e r a l term "paramural body" - r e g a r d l e s s o f t h e i r o r i g i n ; a f u r t h e r d i v i s i o n o f paramural b o d i e s i n t o two c l a s s e s a c c o r d i n g t o t h e i r d e r i v a t i o n - -"lomasomes", d e r i v e d from c y t o p l a s m i c membranes and "p1asma1emmasomes" d e r i v e d e n t i r e l y from t he plasmalemma. By t h i s system the s t r u c t u r e s o b s e r v e d w i t h i n t he c e l l s o f L L h o r d e i would be paramural b o d i e s . S i n c e the o r i g i n o f t h e s e b o d i e s i s unknown f u r t h e r c l a s s i f i c a t i o n c annot be made. T h i s e l e c t r o n m i c r o s c o p e s t u d y o f U_. h o r d e i a t t e m p t e d t o answer t h e q u e s t i o n o f how many n u c l e i per c e l l a r e p r e s e n t . U n f o r t u n a t e l y t h e r e i s s t i l l no d e f i n i t e answer. A c c o r d i n g t o Wang (1934) the c e l l s c o n t a i n one o r s e v e r a l n u c l e i . , In the e n l a r g e d c e l l s , i . e . t h o s e e e l Is w h i c h a c c u m u l a t e c y t o p l a s m and a r e s e a l e d o f f from the empty c o n t r a c t e d c e l l s by s e p t a , she r e p o r t e d one but more o f t e n two n u c l e i , and t h a t t he n u c l e u s o f u n i n u c l e a t e c e l l s can d i v i d e i n such a way t h a t u l t i m a t e l y a l l t h e e n l a r g e d c e l l s c o n t a i n two n u c l e i . B a s i c a l l y t h i s was found t o be the c a s e i n t h i s s t u d y . A r e a s o f some hyphae c o n t a i n e d o n l y one n u c l e u s w h i l e o t h e r s c o n t a i n e d two, as would be e x p e c t e d f o r a d i k a r y o n . Where o n l y one n u c l e u s was p r e s e n t the e n t i r e c e l l c o u l d not always be s e e n , so a second n u c l e u s may have been p r e s e n t e l s e w h e r e i n the c e l l . T h i s i s s u p p o r t e d - b y the' f a c t t h a t c o n j u g a t e n u c l e i have never been r e g u l a r l y o b s e r v e d i n smut f u n g i (C.O. P e r s o n , p e r s o n a l c o m m u n i c a t i o n ) . That the d i k a r y o n s t a t e was seen o n l y i n f r e q u e n t l y may be e x p l a i n e d i n p a r t by t h e unusual r e s u l t s o b t a i n e d from the m a t i n g t e s t . The mutant m y c e l i a l c o l o n y o f U_. h o r d e i seemed t o be a minus (-) m a t i n g type and presumably m o n o k a r y o t i c . However, work done by McLaren (1967) i n d i c a t e s t h a t a c o l o n y can be d i k a r y o t i c but i n c o n s t a n t d i s s o c i a t i o n budding o f f o n l y one m a t i n g t y p e . I f t h i s i s i n f a c t the c a s e then u n i n u c l e a t e d c e l l s would not be e x c e p t i o n a l . Whether o r not the m y c e l i a l c o l o n y i s d e f i n i t e l y d i k a r y o t i c has not been r e s o l v e d c o n c l u s i v e l y . A c c o r d i n g t o D i n o o r and P e r s o n (1969) the growth i n c u l t u r e o f f o r c e d d i k a r y o n s was i n t e r p r e t e d i n terms o f a s s o c i a t i o n - d i s s o c i a t i o n (mono-ka r y o n s t o d i k a r y o n s and r e v e r s e ) . The g e n e r a l u l t r a s t r u c t u r e o f the n u c l e u s i n a p i c a l and d i f f e r e n t i a t e d r e g i o n s o f hyphae was s i m i l a r t o t h a t d e s c r i b e d f o r o t h e r f u n g i ( B r a c k e r , 1967) e x c e p t t h a t the n u c l e i seemed more e l o n g a t e than u s u a l . Such e l o n g a t e n u c l e i have been r e p o r t e d i n P o l y s t i c t u s v e r s i c o l o r ( G i r b a r d t , 1961); Sch i zophy 1 1 urn commune ( V o e l z and N i e d e r p r u e m , 1964), and N'eurospora c r a s s a -( N a m b o o d i r i , 1966). Namboodiri (1966) a s s o c i a t e s t h e e l o n g a t e n u c l e i w i t h r a p i d growth o f the mycelium. N u c l e o l i were o n l y seen o c c a s i o n a l l y i n U_. h o r d e i , but more commonly the n u c l e o p l a s m was u n i f o r m l y dense and g r a n u l a r w i t h no e v i d e n c e o f a n u c l e o l u s . However, n u c l e o l i have b e e n . r e p o r t e d q u i t e f r e q u e n t l y f o r f u n g i f o r some time ( G i r b a r d t , 1958; Edwards e t a 1., 1959; T h y a g a r a j a n , 1963; C o n t i et_ aj_. , 1963; Hawker, 1965)- Perhaps i t was a c a s e o f s e c t i o n i n g i n the c o r r e c t p l a n e i n o r d e r t o see the n u c l e o l u s i n U. h o r d e i . The n u c l e a r membrane i s a t y p i c a l porous d o u b l e membrane ( B r a c k e r , 1967), but somewhat i r r e g u l a r and v a r i a b l e i n d i m e n s i o n s . N u c l e a r pores were o n l y seen i n permanganate f i x e d m a t e r i a l , and were v e r y few i n number. A p a u c i t y o f n u c l e a r pores was a l s o r e p o r t e d f o r t he B a s i d i o m y c e t e Lenz i t e s saep i a r i a (Hyde and Walkinshaw, 1966) but numerous pores have been r e p o r t e d f o r t he D i s c o m y c e t e , Ascodesm i s (Moore, 1962); Pythium debaryanum (Hawker and A b b o t t , 1963b); and Neu r o s p o r a c r a s s a ( N a m b o o d i r i , 1966). G1utara1dehyde-osmiurn t e t r o x i d e f i x a t i o n made the n u c l e a r e n v e l o p e i n LL h o r d e i r a t h e r i n d i s t i n c t , t h e r e f o r e o b s c u r i n g any pores i f they were p r e s e n t . On the o t h e r hand t h e r e may be o n l y a few p o r e s , o r o t h e r v e r y m i n u t e pores beyond t h e r e s o l u t i o n o f the m i c r o s c o p e . I n t e r c o n n e c t i o n s between t h e n u c l e a r e n v e l o p e and the e n d o p l a s m i c r e t i c u l u m have been r e p o r t e d q u i t e e x t e n s i v e l y f o r f u n g i (Edwards and Edwards, 1960; Moore, 1962; Hawker and A b b o t t , 1963b; W e l l s , 1964; Corfman, 1966; N a m b o o d i r i , 1966; C a r r o l l , 1969)- There a r e a l s o some r e p o r t s o f c o n n e c t i o n s between the m i t o c h o n d r i a , plasma membrane and the n u c l e a r e n v e l o p e ( C o n t i e t a 1 . , 1963; T h y a g a r a j a n e_t_ aj_. , 1963)- N e i t h e r i n t e r c o n n e c t i o n s nor b l e b b i n g o f the n u c l e a r e n v e l o p e were o b s e r v e d i n LL h o r d e i . B l e b b i n g o f f o f membranes from the n u c l e a r e n v e l o p e may p o s s i b l y o c c u r ( F i g s . 11 and 12) but t h i s p r o c e s s i s h i g h l y s p e c u l a t i v e . N u c l e i i n permanganate f i x e d m a t e r i a l o f t e n show s t e l 1 a t e - 1 i k e p r o j e c t i o n s (P, F i g s . 11 and 12) from w h i c h membrane o r v e s i c l e b l e b b i n g may o c c u r . However, the e v i d e n c e i s i n c o n c l u s i v e , and s i n c e t he n u c l e a r e n v e l o p e i s f r e q u e n t l y i n d i s t i n c t the e x i s t e n c e o f b l e b b i n g o r i n t e r c o n n e c t i o n s between the n u c l e a r e n v e l o p e and o t h e r o r g a n e l l e s cannot be s e r i o u s l y p r o p o s e d . Stages o f n u c l e a r d i v i s i o n , s p i n d l e a p p a r a t u s , and c e n t r i o l e s were not seen i n t h e hyphae o f U. h o r d e i . A r a t h e r d i f f e r e n t form o f n u c l e a r d i v i s i o n t a k e s p l a c e i n f u n g i ( B a k e r s p i g e l , 1959; B r a c k e r , 1967; G i r b a r d t , 1969; W e l l s , 1969) but n o t h i n g r e s e m b l i n g p o s s i b l e d i v i s i o n s t a g e s was o b s e r v e d . T r u e c e n t r i o l e s as seen in A l b u g o Candida ( B e r l i n and Bowen, 1964b) would not be e x p e c t e d i n U_. h o r d e i because i t does not have a m o t i l e s t a g e . Chromosomes o f f u n g i a r e v e r y d i f f i c u l t t o e l u c i d a t e by e l e c t r o n m i c r o s c o p y , and U_. h o r d e i was no e x c e p t i o n . Chromosomes or s t r u c t u r e s r e s e m b l i n g chromosomes were not seen. However, chromosomes o r at l e a s t d i s t i n c t c h o m a t i n have been o b s e r v e d and s t u d i e d i n some Ascomycetes ( W e l l s , 1969) and some B a s i d i o m y c e t e s ( L u , 1966b; G i r b a r d t , 1969). A l t h o u g h . n o s p i n d l e a p p a r a t u s was se e n , d i s t i n c t m i c r o t u b u l e s were d i s t i n g u i s h e d i n U_. h o r d e i i n the a p i c a l and d i f f e r e n t i a t e d a r e a s o f the hyphae. These were most o f t e n found c l o s e t o t h e o u t s i d e o f the n u c l e a r e n v e l o p e , r u n n i n g p a r a l l e l t o the long a x i s o f t h e n u c l e u s . Some were seen i n t h e c y t o p l a s m near the plasma membrane. M i c r o t u b u l e s i n f u n g i were f i r s t r e p o r t e d by S h a t l a et_ aj_. (1966) and have s i n c e been r e p o r t e d by o t h e r s ( W i l l i a m s and Yukawa, 1967), but no p a r t i c u l a r f u n c t i o n has been a t t r i b u t e d t o them. Newcomb (1969) i n a r e v i e w a r t i c l e d i s c u s s e d t he p o s s i b l e r o l e s o f m i c r o t u b u l e s o t h e r than t h e i r f u n c t i o n s i n c i l i a , f l a g e l l a , and the mi t o t i c s p i n d 1 e . A l t h o u g h t h e r e appears t o be no e v i d e n c e o r s u p p o r t f o r t h e v i e w t h a t m i c r o t u b u l e s a d j a c e n t t o the w a l l p a r t i c i p a t e i n t h e e n z y m a t i c s y n t h e s i s o f the c e l l u l o s e m i c r o f i b r i l s , i t i s g e n e r a l l y agreed t h a t they a r e i n v o l v e d " d i r e c t l y o r i n d i r e c t l y " i n o r i e n t i n g the m i c r o f i b r i l s as they a r e d e p o s i t e e ) , a t l e a s t i n h i g h e r p l a n t s . Newcomb f u r t h e r p o i n t s out two i n t e r r e l a t e d p r o p e r t i e s o f m i c r o t u b u l e s . F i r s t , " t h e y seem t o p l a y a d i r e c t i v e r o l e i n m o r p h o g e n e s i s , e i t h e r by c o n t r o l l i n g t he movement and p o s i t i o n i n g o f o t h e r c e l l u l a r s t r u c t u r e s o r by s e r v i n g as c y t o s k e l e t a l a g e n t s t o b r i n g about and m a i n t a i n c e l l symmetry. Second, as h i g h l y l a b i l e s t r u c t u r e s , t h a t a r e b e l i e v e d t o e x i s t i n dynamic e q u i l i b r i u m w i t h a pool o f r e a d i l y a v a i l a b l e s u b u n i t s , they can be p o l y m e r i z e d o r depo1ymerized r a p i d l y a t d i f f e r e n t i n t r a c e l l u l a r l o c i as c o n d i t i o n s change d u r i n g growth and d i f f e r e n t i a t i o n " . In the a p i c a l and d i f f e r e n t i a t e d r e g i o n s o f the m y c e l i a l c o l o n y o f U_. h o r d e i m i t o c h o n d r i a a r e v e r y numerous. T h i s c o n f i r m s Wang's ( 1 9 3 4 ) s t a t e m e n t t h a t m u l t i p l i c a t i o n o f m i t o c h o n d r i a i s i n t e n s e d u r i n g development o f m y c e l i u m . These m i t o c h o n d r i a cannot r e a l l y be d e s c r i b e d as t y p i c a l f o r f u n g i , a t l e a s t not as r e p o r t e d f o r most f u n g i ( B l o n d e l and T u r i a n , 19 6 0 ; Werner et_ aj_. , 1 9 6 4 a , b; B r a c k e r , 1 9 6 7 ) - Fungi u s u a l l y have p l a t e - l i k e c r i s t a e ( W e l l s , 1 9 6 4 a ; B r a c k e r , 1 9 6 7 ) " but they a r e g e n e r a l l y not o r i e n t e d p a r a l l e l t o the long a x i s o f the m i t o c h o n d r i o n as they a r e i n U_. horde i . The f o l l o w i n g f u n g i have p a r a l l e l o r i e n t a t i o n o f c r i s t a e a l t h o u g h t h i s arrangement i s n ' t a lways c h a r a c t e r i s t i c : Saccharomyces ( H i r a n o and L i n d e g r e n , 1 9 6 1 ; Osumi, 1 9 6 5 ) ; Neurospora c r a s s a ( S h a t k i n and Tatum, 1 9 5 9 ; Lowry and Sussman, 1 9 6 4 ) ; and P u c c i n i a s o r g h i (Van Dyke and Hooker, 1 9 6 9 ) • The m i t o c h o n d r i a l o u t e r membrane i n U_. h o r d e i i s u s u a l l y t h i c k e r than the i n n e r one. T h i s i s a uni q u e s i t u a t i o n because f o r most o r g a n i s m s t h e r e v e r s e i s t r u e , the o u t e r membrane i s s l i g h t l y t h i n n e r than the i n n e r one ( P a r s o n s e t aj_. , 1 9 6 6 ) . The most unusual f e a t u r e o f the m i t o c h o n d r i a o f U_. h o r d e i i s the s e r i e s o f changes t h a t seem t o t a k e p l a c e w i t h i n them i n the d i f f e r e n t i a t e d r e g i o n s o f hyphae. The changes appear t o be i n i t i a t e d by a s l i g h t b u l g e on one s i d e c o n t a i n i n g an e l e c t r o n opaque a r e a . A r m e n t r o u t et_ aj_. ( 1 9 6 8 ) r e p o r t e d t h a t m i t o c h o n d r i a a r e v e r y p l e o m o r p h i c , and f i l a m e n t o u s m i t o c h o n d r i a o f t e n have s w o l l e n o r d i l a t e d a r e a s . However, t h e m i t o c h o n d r i a l b u l g e o f \L' h o r d e i o c c u r s o n l y on one s i d e . A l a r g e i n t e r n a l body was r e p o r t e d i n y e a s t m i t o c h o n d r i a ( L i n d e g r e n e_t_ aj_. , 1 9 6 5 ) , w h i c h i s s i m i l a r t o the e l e c t r o n opaque a r e a i n the m i t o c h o n d r i a o f U_. h o r d e i . T h i s i s p r o b a b l y not an it w i t h the e x c e p t i o n o f t h e t u b u l a r c r i s t a e i n t h e Oomycetes. homologous s t r u c t u r e because a l l c u l t u r e s of y e a s t i n w h i c h t h e s e b o d i e s were found had a l y t i c h i s t o r y , so the b o d i e s a r e p r o b a b l y r e l a t e d i n some way t o the v i r a l i n f e c t i o n . W i t h i n the b u l g e o f LL h o r d e i m i t o c h o n d r i a , c o i l s o f c o n c e n t r i c l a m e l l a e form, and can d e v e l o p q u i t e e x t e n s i v e l y (C, F i g . 21). When t h e s e c o i l s o f c o n c e n t r i c l a m e l l a e have formed they appear t o be t a k e n i n t o v a c u o l e s by i n v a g i n a t i o n o f t h e v a c u o l e membrane ( F i g s . 2Q and 23). Then f u r t h e r changes i n the c o i l s o f l a m e l l a e t a k e p l a c e w i t h i n the v a c u o l e ; t h e s e changes w i l l be d i s c u s s e d p r e s e n t l y . To the b e s t o f the a u t h o r ' s knowledge t h e r e a r e no o t h e r r e p o r t s i n the l i t e r a t u r e r e v i e w e d , t h a t d e s c r i b e such a phenomenon f o r m i t o c h o n d r i a . There a r e many r e p o r t s o f a n o m a l i e s of m i t o c h o n d r i a i n both a n i m a l s and o t h e r p l a n t s ( H i r a n o and L i n d e g r e n , 1963; Goyer and K r a l l , 1969; Bowers and K o r n , 1969; Le Beux e t a l . , 1-969) bat t h e s e a n o m a l i e s a r e u s u a l l y due t o an abnormal e n v i r o n m e n t o f t h e o r g a n i s m , o r t o an abnormal n u t r i t i o n a l c i r c u m s t a n c e . The c o n c e n t r i c a r r a y s r e s e m b l i n g m y e l i n f i g u r e s found i n r a t l i v e r m i t o -chond r i a (Le Beux et_ a_l_. , 1969) a r e q u i t e s i m i l a r t o t h o s e of U_. horde i . Perhaps i n U_. h o r d e i t h i s p e c u l i a r i t y o f t h e m i t o c h o n d r i a r e l a t e s t o the f a c t t h a t the' m y c e l i a l c o l o n y b e i n g s t u d i e d i s a mutant. That the changes o b s e r v e d i n the m i t o c h o n d r i a c o u l d be the r e s u l t o f f i x a t i o n a r t i f a c t s has been r u l e d out because they a r e seen f r e q u e n t l y i n both g l u t a r a l d e h y d e -osmium t e t r o x i d e , and permanganate f i x e d m a t e r i a l , and because i n t e r m e d i a t e s t e p s i n the changes have been o b s e r v e d . I t i s b e l i e v e d t h a t the f o r m a t i o n o f c o n c e n t r i c l a m e l l a e by t h e m i t o c h o n d r i a and subsequent r e l e a s e o f t h e s e l a m e l l a e i n t o v a c u o l e s may be a p a r t o f the d e g e n e r a t i o n p r o c e s s i n t h e hyphae, s i n c e t h e r e i s some r e d u c t i o n i n the number of c r i s t a e as the c o n c e n t r i c l a m e l l a e form; and t h i s phenomenon was most f r e q u e n t l y o b s e r v e d i n o l d e r a r e a s of hyphae. P o s s i b l y the c r i s t a e a r e t r a n s f o r m e d i n t o the membranes o f the c o n c e n t r i c l a m e l l a e . Le Beux e t a l . (1969) 29 a l s o d i s c u s s e d the ide a t h a t the m y e l i n f i g u r e s o b s e r v e d i n r a t l i v e r m i t o c h o n d r i a may be d e g e n e r a t i v e and t h e r e f o r e r e p r e s e n t a d e g e n e r a t i o n o f the c e l l u l a r l i p o p r o t e i n framework. In t h i s c a s e such d e g e n e r a t i o n was induced by s p e c i f i c d i e t a r y c i r c u m s t a n c e s . Perhaps the m i t o c h o n d r i a l c o n c e n t r i c l a m e l l a e f o r m a t i o n o f U. h o r d e i i s an energy p r o d u c i n g p r o c e s s . No t e s t s were made t o d e t e r m i n e whether or not t h e m i t o c h o n d r i a a r e s t i l l f u n c t i o n a l w h i l e t h e s e changes a r e t a k i n g p l a c e . V e s i c l e s o r s m a l l v a c u o l e s a r e q u i t e numerous i n young, a c t i v e l y g r o w i n g hyphae. From t h e i r s i z e and appearance they seem t o f i t i n t o t he c a t e g o r y o f " m i c r o b o d i e s " as d e s c r i b e d by B r a c k e r (1967)• In h i s d e f i n i t i o n m i c r o b o d i e s appear "as s m a l l v e s i c l e s , l e s s than 1 u i n d i a m e t e r , t h a t a r e p h y s i c a l l y a s s o c i a t e d w i t h the e n d o p l a s m i c r e t i c u l u m and may c o n t a i n i n c l u s i o n s " . S i n c e t h e s e v e s i c l e s o r m i c r o b o d i e s a r e always seen i n c l o s e a s s o c i a t i o n w i t h the e n d o p l a s m i c r e t i c u l u m ( v, F i g . 17), i t i s thought t h a t p o s s i b l y t h e e n d o p l a s m i c r e t i c u l u m g i v e s r i s e t o them by d i l a t i o n o f the c i s t e r n a e and b l e b b i n g o f f . T h i s p r o c e s s i s su g g e s t e d by f i g u r e 14 ( a r r o w ) . Both v e s i c l e s and e n d o p l a s m i c r e t i c u l u m a r e more numerous i n d i f f e r e n t i a t e d r e g i o n s . The ide a o f the e n d o p l a s m i c r e t i c u l u m p r o d u c i n g v e s i c l e s o r s m a l l v a c u o l e s i s not a new one. I t was proposed by P o r t e r and Machado (i960) and by Buvat (1963). P o r t e r and Machado (i960) s u g g e s t e d t h a t a s p e c i a l i z e d t y p e o f e n d o p l a s m i c r e t i c u l u m g i v e s r i s e t o v e s i c l e s and v a c u o l e s . S i n c e t h e n , more e v i d e n c e has a c c u m u l a t e d t o s u p p o r t the i d e a o f v e s i c l e and v a c u o l e f o r m a t i o n from the e n d o p l a s m i c r e t i c u l u m ( B e r l i n and Bowen, 1964a; Werner et^ aj_. , 1966) . Namboodiri (1966) r e p o r t e d i n s t a n c e s i n Ne u r o s p o r a c r a s s a mycelium where the ends o f t h e e n d o p l a s m i c r e t i c u l u m e n l a r g e t o form v e s i c l e s . A system o f v e s i c l e s produced by the e n d o p l a s m i c r e t i c u l u m was o b s e r v e d i n Phycomyces b1akes1eeanus, F u s a r i u m culmorum and C o p r i n u s lagopus (Marchant e t a l . , 1967)- Peat and Banbury (1967) r e p o r t e d t h r e e types o f v e s i c l e a s s o c i a t e d w i t h the en d o p l a s m i c r e t i c u l u m - thos e w i t h f a i n t l y g r a n u l a r c o n t e n t s , m u l t i -v e s i c u l a r b o d i e s , and d i s t e n d e d i s o l a t e d s t r a n d s o f e n d o p l a s m i c r e t i c u l u m , i n Phycomyces. A l t h o u g h the f u n c t i o n o f such v e s i c l e s i s p r o b l e m a t i c , B r a c k e r (1967) s t a t e d t h a t t h e i r c h a r a c t e r i s t i c s a r e c o n s i s t e n t w i t h p r o t e i n s t o r a g e o r enzyme c o m p a r t m e n t a 1 i z a t i o n . T h i s seems f e a s i b l e i n the c a s e o f U_. h o r d e i . The numerous ribosomes t h a t a r e always a s s o c i a t e d w i t h the e n d o p l a s m i c r e t i c u l u m c o u l d s y n t h e s i z e the enzymes w h i c h would then be c o n c e n t r a t e d i n the c i s t e r n a e of the e n d o p l a s m i c r e t i c u l u m , and bl e b b e d o f f as v e s i c l e s o r m i crobod i es.. Hawker and Gooday (1969) s t u d i e d d i s s o l u t i o n o f the a p i c a l w a l l s o f the progametang i a o f Rh i zopus sexua l i s . They d e s c r i b e d v e s i c l e s and 1omasome-1ike masses i n p o c k e t s next t o the c e l l w a l l , formed by the w r i n k l i n g o f the plasma membrane. T h e i r o b s e r v a t i o n s s u g g e s t e d t h a t the v e s i c l e s were d e r i v e d from the e n d o p l a s m i c r e t i c u l u m , and p r o b a b l y c o n t a i n e d enzymes a s s o c i a t e d w i t h w a l l s y n t h e s i s o r w a l l d e g r a d a t i o n , depending on the l o c a l i z e d r e g u l a t i n g f a c t o r . In the c a s e o f Rh i zopus sexua1i s t h e v e s i c l e s seemed t o c o n t a i n enzymes d i r e c t e d towards w a l l d i s s o l u t i o n . Wang's (1934) r e p o r t t h a t young m y c e l i a l f i l a m e n t s o f U_. horde i c o n t a i n a number of s m a l l v a c u o l e s and t h a t w i t h a g i n g o f the mycelium the v a c u o l e s become b i g g e r , was c o n f i r m e d by t h i s s t u d y . V a c u o l e s were found t o c o n t a i n a v a r i e t y o f m a t e r i a l r a n g i n g from s o l i d e l e c t r o n dense m a t e r i a l t o c o n c e n t r i c l a m e l l a e o r tubu 1 a r b o d i e s . Of g r e a t e s t i n t e r e s t a r e the v a c u o l e s c o n t a i n i n g the c o n c e n t r i c l a m e l l a e o r t u b u l a r b o d i e s . These s t r u c t u r e s v e r y much resemble the c o i l s formed by the m i t o c h o n d r i a . • S i m i l a r c o i l s o r m y e l i n f i g u r e s have been r e p o r t e d r e c e n t l y i n o t h e r f u n g i ( B u c k l e y et_ aj_. , 1966; S h a t l a et_ aj_. , 1966; Thomas and I s s a c , 1967; Kunoh and A k a i , 1969; Van Dyke and Hooker, 1969)- The m y e l i n f i g u r e s , d e n s e l y s t a i n i n g l o o p s and c i r c u l a r l a m i n a t e d s t r u c t u r e s i n B o t r y t i s  c i nerea c o n i d i a ( B u c k l e y et aj_. , 1 9 6 6 ) were b e l i e v e d t o r e p r e s e n t h y d r a t e d p h o s p h o l i p i d , and were taken t o i n d i c a t e d e g e n e r a t i o n when found i n the c y t o p l a s m . When such s t r u c t u r e s were o b s e r v e d i n the v a c u o l e s o f g e r m i n a t i n g s p o r e s they were i n t e r p r e t e d as s t a g e s i n the m o b i l i z a t i o n o f r e s e r v e s . S i m i l a r s t r u c t u r e s i n P u c c i n i a s o r g h i (Van Dyke and Hooker, I 9 6 9 ) were i n t e r p r e t e d as t u b u l a r e x t e n s i o n s o f t h e f u n g a l plasma membrane. In the c e l l u l a r s l i m e molds D i c t y o s t e l i u r n d i s c o i d e u m and Po 1ysphondy 1iurn  pa 1 1 i dum ( H o h l , 1 9 6 5 ) w h i c h a r e p r e d a t o r y upon b a c t e r i a , systems o f c o n c e n t r i c l a m e l l a e appear i n the f o o d v a c u o l e s . T h i s s t u d y r e v e a l e d t h a t t h e s e l a m e l l a e do not ^represent o r i g i n a l membranes o f the i n g e s t e d b a c t e r i a but a r e membranes formed as a r e s u l t o f e n z y m a t i c r e a c t i o n s i n v o l v e d i n d i g e s t i on. I t i s g e n e r a l l y h e l d t h a t .large v a c u o l e s form t h r o u g h f u s i o n o f s m a l l e r v a c u o l e s or v e s i c l e s . E v i d e n c e i n d i c a t e s t h a t t h i s i s the c a s e i n LL h o r d e i ( F i g s . 17 and 2 3 ) . T h e r e f o r e , i f the v e s i c l e s a r e i n f a c t enzyme compartments, the v a c u o l e s w h i c h a r i s e as a r e s u l t o f f u s i o n o f v e s i c l e s would be r i c h i n enzymes. V a c u o l e s c o n t a i n i n g numerous enzymes i n a n i m a l c e l l s a r e known as lysosomes. S i m i l a r v a c u o l e s i n h i g h e r p l a n t s and f u n g i have been proposed as the lysosomes o f p l a n t s by M a t i l e and Wiemken ( 1 9 6 7 ) and M a t i l e ( 1 9 6 8 a , b, c ) . M a t i l e ' s work i n v o l v e d i s o l a t i o n o f v a c u o l e s from y e a s t ( M a t i l e and Wiemken, 1 9 6 7 ) and from r o o t t i p c e l l s o f c o r n s e e d l i n g s ( M a t i l e , 1 9 6 8 a ) . B i o c h e m i c a l a s s a y s of the c o n t e n t s of t h e s e v a c u o l e s i n d i c a t e d the p r e s e n c e o f c o n s i d e r a b l e numbers o f h y d r o l y t i c enzymes. These r e s u l t s l e d M a t i l e t o c o n c l u d e t h a t such v a c u o l e s r e p r e s e n t l y s o s o m e s . A more r e c e n t s t u d y o f u l t r a s t r u c t u r a l l o c a l i z a t i o n o f a c i d p h o s p h a t a s e i n c u l t u r e d c e l l s o f Daucus c a r o t a ( H a l p e r i n , 1 9 6 9) r e p o r t e d a c i d p h o s p h a t a s e a c t i v i t y i n v a c u o l e s and d i c t y o s o m e - d e r i v e d v e s i c l e s . In U. h o r d e i v a c u o l e s a r e seen t o f u s e w i t h the plasma membrane and t o a p p a r e n t l y r e l e a s e t h e i r c o n t e n t s i n t o the space between the c e l l w a l l and the plasma membrane. M a t i l e (1965) and M a t i l e et_ aj_. (1965) r e p o r t e d t h a t p r o t e a s e s , s t o r e d i n t r a c e 11u1 ar1y i n membrane bound v e s i c l e s i n Neurospora c r a s s a , c r o s s e d the plasma membrane as i n t a c t p a r t i c l e s by means o f i n v a g i n a t i o n s o f the plasma membrane. These p r o t e a s e p a r t i c l e s were b e l i e v e d t o o r i g i n a t e from the e n d o p l a s m i c r e t i c u l u m . M a t i l e (19&5) a l s o m e ntioned t h a t an a m i n o p e p t i d a s e i s l o c a l i z e d o u t s i d e the plasma membrane o f the Neurospora c r a s s a m y c e l i a l c e l l s , p r o b a b l y between the plasma membrane and the c e l l w a l l . The whole sequence o f the o r i g i n and development of lysosomes i n c o r n r o o t t i p c e l l s has now been worked out by M a t i l e and Moor (1968). They have proposed the f o l l o w i n g g e n e s i s o f v a c u o l e s ( l y s o s o m e s ) : (a) F o r m a t i o n o f s m a l l e n d o p l a s m i c r e t i c u 1 u m - d e r i v e d v e s i c l e s ( p r o v a c u o 1 e s ) ; (b) f u s i o n o f p r o v a c u o l e s r e s u l t i n g i n the f o r m a t i o n o f s m a l l v a c u o l e s , f o l l o w e d by the f u s i o n ' a n d e x p a n s i o n o f v a c u o l e s ; (c) i n c o r p o r a t i o n o f l a r g e d i c t y o s o m e - d e r i v e d v e s i c l e s i n t o v a c u o l e s by i n v a g i n a t i o n of the t o n o p l a s t ; (d) i n v a g i n a t i o n o f the t o n o p l a s t r e s u l t i n g i n t he i n c o r p o r a t i o n o f c y t o p l a s m i c m a t e r i a l i n t o v a c u o l e s . P r o v a c u o l e s a r e shown t o be p r i m a r y lysosomes w i t h t h e i r h y d r o l a s e s a r i s i n g from the en d o p l a s m i c r e t i c u l u m . On the b a s i s o f the e v i d e n c e o b t a i n e d from t h i s s t u d y o f U_. h o r d e i , and from s u p p o r t i n g i n f o r m a t i o n i n the l i t e r a t u r e , t he f o l l o w i n g h y p o t h e s i s i s s u g g e s t e d as an e x p l a n a t i o n f o r the sequence o f e v e n t s t h a t a r e seen to o c c u r i n the mutant m y c e l i a l c o l o n y o f U. h o r d e i : ( l ) V a r i o u s enzymes a r e s y n t h e s i z e d on the ribosomes i n the hyphal c y t o p l a s m ; (2) t h e s e .enzymes then a c c u m u l a t e i n the c i s t e r n a e o f t h e e n d o p l a s m i c r e t i c u l u m and a r e packaged i n t o v e s i c l e s ( p r o v a c u o l e s ) t o be ble b b e d o f f by t h e e n d o p l a s m i c r e t i c u l u m ; (3) the v e s i c l e s ( p r o v a c u o l e s ) f u s e t o form s m a l l v a c u o l e s w h i c h i n c o r p o r a t e c y t o p l a s m i c m a t e r i a l , and o t h e r v a c u o l e s c o n t a i n i n g c o n c e n t r i c l a m e l l a e c o i l s produced by the m i t o c h o n d r i a , by i n v a g i n a t i o n o f the v a c u o l e membrane; (4) f u r t h e r f u s i o n o f v a c u o l e s u n t i l a t some p o i n t , the r e s u l t i n g v a c u o l e s f u s e w i t h the plasma membrane and r e l e a s e t h e i r c o n t e n t s i n t o the space between the w a l l and the plasma membrane. W i t h i n the v a c u o l e s c o n t a i n i n g c o n c e n t r i c l a m e l l a e complexes e n z y m a t i c a c t i v i t y p r o b a b l y c o n v e r t s t h e c o n c e n t r i c l a m e l l a e t o the t u b u l a r b o d i e s . The n a t u r e o f t h e m a t e r i a l r e l e a s e d i s unknown. I t may r e p r e s e n t a means of s e c r e t i n g e x t r a c e l l u l a r enzymes (Bech-Hansen, 1970), or a c o n t r i b u t i o n t o c e l l w a l l s y n t h e s i s , o r a means o f g e t t i n g r i d o f unwanted m a t e r i a l o r p r o d u c t s r e s u 1 t i n g . f r o m the d e g e n e r a t i v e p r o c e s s . The absence of a d i c t y o s o m e i n U_. horde i i s c o n s i s t e n t w i t h the f a c t t h a t most p l a n t pathogens a r e i n groups o f f u n g i t h a t do not p o s s e s s t h i s o r g a n e l l e ( B r a c k e r , 1967, 1968). I t i s assumed t h a t the f u n g a l G o l g i a p p a r a t u s p a r t i c i p a t e s i n some a s p e c t o f s e c r e t i o n . T h e r e f o r e , " t h e a p p a r e n t absence o f d i c t y o s o m e s i n so many f u n g i r a i s e s the q u e s t i o n o f what c e l l component i f any, c a r r i e s out the e x p e c t e d f u n c t i o n s o f d i c t y o s o m e s i n c e l l s where none a r e p r e s e n t " ( B r a c k e r , 1967)- B r a c k e r s t a t e d t h a t a membrane-bound s t r u c t u r e c a p a b l e o f p a c k a g i n g m a t e r i a l s w i t h i n a membrane f o r t r a n s p o r t would be l o g i c a l f o r t h i s r o l e . In LL horde i i t seems t h a t the e n d o p l a s m i c r e t i c u l u m i s a b l e t o c a r r y out the r o l e o f s e c r e t i o n . In a n o t h e r Bas<diomycete l a c k i n g a d i c t y o s o m e , Armi11 a r i a , B r a c k e r (1969) has r e p o r t e d an a s s o c i a t i o n between the e n d o p l a s m i c r e t i c u l u m and c i s t e r n a l e l e m e n t s t h a t b l e b v e s i c l e s . Septa have been s t u d i e d i n g r e a t d e t a i l i n f u n g i (Moore and M c A l e a r , 1962a; B r a c k e r and B u t l e r , 1962, 1963, 1964; W i l s e n a c h and K e s s e l , 1965a; W e l l s , 1965; R e i c h l e and A l e x a n d e r , 1965; F r a n k , 1967; E h r l i c h and E h r l i c h , 1968). D i f f e r e n t t y p e s o f s e p t a a r e c h a r a c t e r i s t i c f o r t h e d i f f e r e n t groups o f f u n g i . These have been e x t e n s i v e l y r e v i e w e d by Hawker (1965) and B r a c k e r (1967)- The s e p t a o f U_. h o r d e i a r e s i m p l e and c o m p l e t e . A c c o r d i n g t o B r a c k e r (1967) t h i s t y p e o c c u r s m a i n l y i n the Phycomycetes, Hemiascomycetes and some D e u t e r o m y c e t e s , and i n c e l l s t h a t a r e i n j u r e d or a d j a c e n t t o e v a c u a t e d c e l l s . No pores o f any k i n d were o b s e r v e d i n U_. h o r d e i s e p t a . Plasmodesmata (Hawker and Gooday, 1966; K i r k and S i n c l a i r , 1966) and micropores., (Hashimoto et_ aj_. , 1964) have been r e p o r t e d f o r o t h e r f u n g i , but n e i t h e r o c c u r i n U_. h o r d e i . S i n c e the hyphal s e p t a e o f Lh horde i o n l y o c c u r next t o o l d , d e g e n e r a t e , o r e v a c u a t e d r e g i o n s , i t i s b e l i e v e d t h a t the r o l e of septum f o r m a t i o n i s a p r o t e c t i v e one, t o s e a l o f f the a c t i v e l y g rowing r e g i o n s from the i n j u r e d or d e g e n e r a t e p a r t s , s i m i l a r t o the p l u g g i n g mechanism i n porous s e p t a (Hawker, 1965; B r a c k e r , 1967)-Growth o f the i n f e c t i v e d i k a r y o t i c hyphae ( i . e . " s u c h f a d e n " ) i s by hyphal e l o n g a t i o n , w i t h c y t o p l a s m " f l o w i n g " t o the t i p , o c c a s i o n a l l y s e a l e d o f f from b e h i n d by septum f o r m a t i o n (C.O. P e r s o n , p e r s o n a l c o m m u n i c a t i o n ) . B r a n c h i n g u s u a l l y o c c u r s a t t h i s septum, so t h e septum may i n i t i a t e b r a n c h i n g . T h i s s t u d y of the mutant m y c e l i a l c o l o n y o f U_. h o r d e i i s by no means c o m p l e t e . The f a c t t h a t the m y c e l i a l c o l o n y i s a mutant s h o u l d be kept i n mind when i n t e r p r e t i n g the f u n c t i o n o f any u n u s u a l hyphal s t r u c t u r e s such as the c o n c e n t r i c l a m e l l a e o f the m i t o c h o n d r i a and the paramural b o d i e s . The f i x a t i o n seems t o be q u i t e adequate but i t c o u l d be improved t h r o u g h f u r t h e r e x p e r i m e n t a t i o n w i t h t h e t e c h n i q u e s used. To d e t e r m i n e whether the f o r m a t i o n of c o n c e n t r i c l a m e l l a e by the m i t o c h o n d r i a and t h e i r subsequent t r a n s f e r t o v a c u o l e s i s p a r t o f a d e g e n e r a t i o n p r o c e s s , c o l o n i e s would have t o be s t u d i e d a t e a r l i e r and l a t e r s t a g e s of g r o w t h . A l s o t o t e s t whether t h e m i t o c h o n d r i a a r e f u n c t i o n a l d u r i n g the c o n c e n t r i c l a m e l l a e f o r m a t i o n , t h e c o l o n i e s c o u l d be exposed t o t e l l u r i t e o r t e t r a z o l i u m s a l t s ( B i s a l p u t r a e t a l . , 1 9 6 9 ) f o r a t i m e p r i o r t o f i x a t i o n . A b l a c k p r e c i p i t a t i o n of t h e s e s u b s t a n c e s w i t h i n the m i t o c h o n d r i a would i n d i c a t e t h a t the m i t o c h o n d r i a a r e f u n c t i o n a l . To t e s t M a t i l e ' s i d e a ( M a t i l e , 1968a, b, c; M a t i l e and Moor, 1968) t h a t v a c u o l e s i n p l a n t s a r e comparable t o lysosomes i n a n i m a l s , v a c u o l e s c o u l d be i s o l a t e d from U_. h o r d e i hyphae u s i n g M a t i l e ' s t e c h n i q u e , and t h e i r c o n t e n t s a s s a y e d f o r a c i d p h o s p h a t a s e and o t h e r h y d r o l y t i c enzymes. A l s o u 1 1 r a s t r u c t u r a 1 a u t o r a d i o g r a p h y c o u l d be done t o t r a c e the o r i g i n and f i n a l d e s t i n a t i o n o f the c o n t e n t s o f the v a c u o l e s . Some h i s t o c h e m i s t r y might be t r i e d t o d e t e r m i n e whether g l y c o g e n i s a c t u a l l y p r e s e n t . S i n c e i t i s the d i k a r y o t i c s t a g e of U. h o r d e i w h i c h i s i n f e c t i v e , i t would be i n t e r e s t i n g t o know whether t h i s mutant m y c e l i a l c o l o n y i s a l s o i n f e c t i v e . T h i s e x p e r i m e n t i s p r e s e n t l y under way but the r e s u l t s a r e not y e t c o m p l e t e . B a r l e y seeds have been i n o c u l a t e d w i t h the f o l l o w i n g (a) m y c e l i a l c o l o n y , s e l f e d (b) m y c e l i a l c o l o n y X 1^ m a t i n g t y p e (c) m y c e l i a l c o l o n y X ma t i n g t y p e . The seeds a r e now grow i n g i n a greenhouse. No smut appears t o be p r e s e n t i n t he f i r s t t i l l e r s o f t h e p l a n t s , but t h e f i n a l r e s u l t s w i l l not be known u n t i l t h e second t i l l e r s a r e formed. SUMMARY The f i l a m e n t o u s hyphae of the mutant m y c e l i a l c o l o n y o f Ust i 1 ago h o r d e i ( P e r s . ) L a g e r h . , s t u d i e d w i t h l i g h t and e l e c t r o n m i c r o s c o p e s , c o n s i s t o f v e r y long c e l l s , 2 . 0 t o 5 - 0 y wide and 11 t o h0 y long w i t h s e p t a o c c u r r i n g a t i r r e g u l a r i n t e r v a l s . A s i n g l e l a y e r e d f i b r i l l a r w a l l s u r r o u n d s t h e hyphae. In a p i c a l r e g i o n s , i n c l u s i o n s a r e p r e s e n t w i t h i n the w a l l i n p l a c e s a l o n g the hypha. The d e n s e l y g r a n u l a r c y t o p l a s m bound by a c o n v o l u t e d plasma membrane c o n t a i n s numerous f r e e r i b o s o m e s ; one o r two n u c l e i , m i t o c h o n d r i a w i t h p l a t e - l i k e c r i s t a e , v a c u o l e s , v e s i c l e s and smooth s u r f a c e d e n d o p l a s m i c r e t i c u l u m . L i p i d , the major s t o r a g e p r o d u c t , i s p r e s e n t i n the a p i c a l r e g i o n s and, i n l e s s e r amounts, i n the d i f f e r e n t i a t e d a r e a s . I t i s ab s e n t from d e g e n e r a t e d r e g i o n s . Some g l y c o g e n may be p r e s e n t i n the d i f f e r e n t i a t e d a r e a s , but t h i s i s u n c e r t a i n . The e l o n g a t e n u c l e i a r e bound by an u n d u l a t i n g d o u b l e membrane w h i c h c o n t a i n s v e r y few n u c l e a r p o r e s . A d e n s e l y g r a n u l a r n u c l e o l u s i s o c c a s i o n a l l y v i s i b l e i n the l e s s g r a n u l a r n u c l e o p l a s m . M i c r o t u b u l e s a r e f r e q u e n t l y seen i n the c y t o p l a s m o f a p i c a l and d i f f e r e n t i a t e d hyphal r e g i o n s next t o t h e n u c l e a r e n v e l o p e o r near t h e plasma membrane. Dictyosomes and lomasomes a r e not p r e s e n t . E l o n g a t e m i t o c h o n d r i a w i t h p l a t e - l i k e c r i s t a e a r e abundant i n the hyphal c e l l s o f a p i c a l and d i f f e r e n t i a t e d a r e a s . The p l a t e - l i k e c r i s t a e l i e p a r a l l e l t o the long a x i s o f t h e m i t o c h o n d r i o n . The o u t e r m i t o c h o n d r i a l membrane i s u s u a l l y s l i g h t l y t h i c k e r than the i n n e r one. In some m i t o c h o n d r i a i n mature c e l l s , c o n c e n t r i c l a m e l l a e form w i t h i n a b u l g e w h i c h p r o j e c t s from 37 the o u t e r membrane on one s i d e of the m i t o c h o n d r i a l e n v e l o p e . V a c u o l e s seem to e n g u l f t h e s e c o n c e n t r i c l a m e l l a e and t r a n s f o r m them i n t o t u b u l a r b o d i e s . These v a c u o l e s e v e n t u a l l y f u s e w i t h the plasma membrane and e x t r u d e t h e i r c o n t e n t s i n t o the space between the w a l l and the plasma membrane. I t i s not known whether t h i s i s a d e g e n e r a t i v e p r o c e s s or an energy p r o d u c i n g p r o c e s s . No r e p o r t of t h i s t y p e o f p r o c e s s was found i n the l i t e r a t u r e r e v i e w e d . V e s i c l e s and v a c u o l e s o f v a r i o u s s i z e s a r e p r e s e n t i n the a p i c a l r e g i o n s o f hyphae but a r e more numerous i n d i f f e r e n t i a t e d and d e g e n e r a t e a r e a s . E n d o p l a s m i c r e t i c u l u m i s s p a r s e i n a p i c a l r e g i o n s , more numerous i n d i f f e r e n t i a t e d a r e a s , and a b s e n t from d e g e n e r a t e r e g i o n s . The v e s i c l e s seem t o a c t as p r o v a c u o l e s w h i c h f u s e t o form l a r g e r v a c u o l e s . V a c u o l e s e n g u l f b o t h o t h e r v a c u o l e s , and the m i t o c h o n d r i a l c o n c e n t r i c l a m e l l a e by i n v a g i n a t i o n of t h e t o n o p l a s t . A l t h o u g h the f u n c t i o n o f the e n d o p l a s m i c r e t i c u l u m i s unknown i n t h i s f u n g u s , t h e r e i s some e v i d e n c e s u g g e s t i n g t h a t the v e s i c l e s a r e formed from t h e e n d o p l a s m i c r e t i c u l u m by b l e b b i n g . I t i s thought t h a t the v a c u o l e s formed by f u s i o n o f t h e s e v e s i c l e s c o n t a i n enzymes produced on the ribosomes and c o n c e n t r a t e d i n the c i s t e r n a e o f the e n d o p l a s m i c r e t i c u l u m . T h e r e f o r e , t h e a c t i o n o f t h e s e v a c u o l e s , i n f u n g i , i s s i m i l a r t o t h a t o f the lysosomes found i n a n i m a l c e l l s . S i m p l e , c o m p l e t e s e p t a o c c u r i r r e g u l a r l y i n the hyphal c e l l s next t o mature, d e g e n e r a t e o r e v a c u a t e d r e g i o n s . T h e i r f u n c t i o n i s thought t o be to s e a l o f f f u n c t i o n a l r e g i o n s and p r o t e c t them from d e g e n e r a t e or i n j u r e d p a r t s . A second p o s s i b l e f u n c t i o n i s t o i n i t i a t e b r a n c h i n g . The p o s s i b l e e x i s t e n c e o f pores i n the s e p t a i s not e x c l u d e d . BIBLIOGRAPHY Acha, I .G., M.J.R. Aguir re , F. Uruburu and J .R . V i l lanueva. 1966. The f ine structure of the Fusarium culmorum conidium. Trans. B r i t . Mycol. Soc. 49: 695-702. ~~ Agar, H.D. and H.C. Douglas. 1955- Electron microscopy of thin sections of yeast. J . Appl. Phys io l . 26: 1393-1396. Agar, H.D. and H.C. Douglas. 1957- Studies on the cyto log ica l structure of yeast: Electron microscopy of thin sect ions. J . B a c t e r i o l . 73_: 365-375. A k a i , S . , M. Fukutomi and H. Kunoh. 1968. An electron microscope study of conidium and hypha of Erysiphe graminis hordei . Mycopathol. Mycol. Appl . 35: 217-222. 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P a r i s 2_48: 274-277-PLATE I L i g h t m i c r o s c o p e p i c t u r e s . F i g u r e 1. G e n e r a l view of the l i v i n g m y c e l i a . Note the v a r i a t i o n i n the d i a m e t e r o f the hypha, and the c r o s s w a l l s . At one p o i n t a branch i s f o r m i n g • ( a r r o w ) . Very few organe11es a r e v i s i b l e . Phase c o n t r a s t . X 1400. F i g u r e 2. Two hyphae. S e v e r a l n u c l e i a r e v i s i b l e and p o s s i b l y some m i t o c h o n d r i a . Note s e a l e d o f f a r e a . P r o p i o n i c - i r o n h a e m a t o x y 1 i n . X 22^0. F i g u r e 3• Hypha w i t h a c o n s t r i c t i o n ( a r r o w ) . Two n u c l e i and numerous m i t o c h o n d r i a a r e p r e s e n t . P r o p i o n i c - i r o n h a e m a t o x y 1 i n . X 22^0. PLATE 2 F i g u r e h. A p i c a l r e g i o n . Nucleus (N) w i t h a d i s t i n c t n u c l e o l u s (NU). The n u c l e a r e n v e l o p e (NE) i s v e r y wavy as i s the plasma membrane (PM). M i c r o t u b u l e s (MT) , ribosomes ( r ) and p o s s i b l y g l y c o g e n (G) a r e p r e s e n t . G l u t - OsO^ f i x a t i o n . X 30,000. F i g u r e 5- Near the a p i c a l t i p . Small v a c u o l e s (V) , some e n d o p l a s m i c r e t i c u l u m (ER) and the u n d u l a t i n g plasma membrane (PM) a r e p r e s e n t . The hypha i s e n c l o s e d by a narrow w a l l (W) wh i c h seems t o have i n c o r p o r a t e d some m a t e r i a l a t v a r i o u s p o i n t s a l o n g i t s l e n g t h ( X ) . G l u t - O s O i f i x a t i o n . X 36,000. PLATE 3 F i g u r e 6. A p i c a l hyphal u l t r a s t r u c t u r e as r e v e a l e d by KMnO, f i x a t i o n : N = n u c l e u s ; NP = n u c l e a r p o r e ; V *=• v a c u o l e ; M ='mi to.chondr i a ; ER = e n d o p l a s m i c r e t i c u l u m ; L = l i p i d . X 11,000. F i g u r e 7- A v e r y f l o c c u l e n t a r e a of the c y t o p l a s m ( F ) . G l u t - OsO, f i x a t i o n . X 36,000. PLATE k F i g u r e 8. KMnO^ f i x e d hypha showing p a r t o f a n u c l e u s ( N), l i p i d (L) and e n d o p l a s m i c r e t i c u l u m (ER') . The plasma membrane (PM) and t h e wal 1 (W) a r e v i s i b l e . X 1 9 , 0 0 0 . F i g u r e 9- An a p i c a l r e g i o n o f a b r a n c h i n g hypha. A n u c l e u s (N) and s e v e r a l m i t o c h o n d r i a (M) a r e o b v i o u s . Note the s e a l e d o f f d e g e n e r a t e a r e a ( a r r o w ) . G l u t - OsO^ f i x a t i o n . X 3 0 , 0 0 0 . PLATE 5 F i g u r e 10. F i gure 11 F i g u r e 12. T y p i c a l e l o n g a t e m i t o c h o n d r i a (N) w i t h p l a t e - l i k e c r i s t a e , p a r a l l e l t o t he long a x i s o f the m i t o c h o n d r i o n (g = g r a n u l e ) . G l u t - OsO, f i x a t i o n . X 3^,000. A n u c l e u s (N) w i t h a number o f unusual p r o t r u b e r a n c e s (P) NP = n u c l e a r p o r e ; NE = n u c l e a r e n v e l o p e X 3 2 , 0 0 0 . KMnO, f i x a t i o n . A s e c t i o n o f a hypha c o n t a i n i n g two n u c l e i (N). Both n u c l e i have • p r o t r u b e r a n c e s .(P) . KMnO^ f i x a t i o n . X 11 ,000. PLATE 6 F i g u r e 13- A d j a c e n t a r e a s o f t h e same hypha. Two r a t h e r i n d i s t i n c t n u c l e i (N) a r e p r e s e n t , s e v e r a l v a c u o l e s (V) c o n t a i n i n g membrane-1amel1ae complexes (ML), and m i t o c h o n d r i a (M). One v a c u o l e a p p ears t o be r e l e a s i n g some m a t e r i a l i n t o the r e g i o n between the plasma membrane and the w a l l (Y - a r r o w ) . G l u t - OsO, f i x a t i o n . X 15,000. PLATE 7 F i g u r e 1k. F i g u r e 15• An o l d e r a r e a f u l l of v a c u o l e s ( V ) , v e s i c l e s (v) e n d o p l a s m i c r e t i c u l u m (ER). G l u t and some X 3 6 , 0 0 0 . OsO, f i x a t i o n . . T y p i c a l e l o n g a t e n u c l e u s (N) i n a r e g i o n o f f l o c c u l e n t c y t o p l a s m . At the bottom r i g h t m i c r o t u b u l e s (MT) can be seen r u n n i n g p a r a l l e l w i t h the n u c l e u s . G l u t - OsO^ f i x a t i o n . X 3 6 , 0 0 0 . PLATE 8 F i g u r e 1 6. F i g u r e 1 7 • Unusual m i t o c h o n d r i a (M) w i t h c r i s t a e f o r m i n g s t r u c t u r e s s i m i l a r t o c o n c e n t r i c l a m e l l a e ( C ) . These m i t o c h o n d r i a a r e i n c l o s e a s s o c i a t i o n w i t h a l a r g e vacuol'e (V) . Ribosomes ( r ) a r e numerous. F i x a t i o n , a m i x t u r e o f G l u t - OsO^ i n c a c o d y l a t e b u f f e r . X 33,000. An a r e a o f numerous v a c u o l e s (V) and m i t o c h o n d r i a appears t o be e n g u l f i n g a n o t h e r v a c u o l e (arrow) e l e c t r o n opaque a r e a (0) i n the c e n t e r (M). Note m i tochond r i on. One v a c u o l e the unusual L = l i p i d ; ER = e n d o p l a s m i c X 61,200. r e t i cu 1 urn: r i b o s o m e s . G l u t - 0s0, f i x a t i o n . PLATE 9 F i g u r e 18. An o b l i q u e s e c t i o n o f a hypha c o n t a i n i n g numerous m i t o c h o n d r i a (M). L = l i p i d . KMnO^ f i x a t i o n . X 21,000. F i g u r e 19-. C o n c e n t r i c l a m e l l a e s t r u c t u r e s (C) w i t h i n b u l g e s i n the o u t e r membranes o f a m i t o c h o n d r i o n (M). G l u t - OsO, f i x a t i o n . X 48,000. PLATE 10 F i g u r e 20. C o n c e n t r i c p r o j e c t i ng X 4 3 , 0 0 0 . l a m e l l a e s t r u c t u r e (C) of a m i t o c h o n d r i o n (M) i n t o a v a c u o l e ( V ) . G l u t - 0s0^ f i x a t i o n . F i g u r e 21 F i g u r e 22. F i g u r e 23. L a r g e , w e l l d e v e l o p e d c o n c e n t r i c l a m e l l a e s t r u c t u r e (C) w i t h i n a b u l g e i n the o u t e r membrane o f a m i t o c h o n d r i o n (M) . Note f l o c c u l e n t c y t o p l a s m ( F ) . (g = g r a n u l e ) . G l u t - 0s0, f i x a t i o n . X 48,000. An a r e a o f f l o c c u l e n t c y t o p l a s m (F) c o n t a i n i n g a c o n c e n t r i c l a m e l l a e s t r u c t u r e (C) i n a s s o c i a t i o n w i t h a membrane bounded t u b u l a r body ( t ) . T u b u l a r bodies" appear t o form from d i s -s o c i a t i o n ' o r b l e b b i n g o f s u r r o u n d i n g membranes ( a r r o w ) . G l u t - OsO^ f i x a t i o n . X 7 2 , 0 0 0 . D i f f u s e m i t o c h o n d r i a (M) a s s o c i a t e d w i t h a l a r g e v a c u o l e ( V ) . The v a c u o l e c o n t a i n s two c o n c e n t r i c l a m e l l a e s t r u c t u r e s (C) and seems t o be e n g u l f i n g a n o t h e r from a c l o s e l y opposed v a c u o l e . S e v e r a l i n v a g i n a t i o n s o f the bounding membrane of the l a r g e v a c u o l e a r e i n d i c a t e d by the a r r o w s . A t u b u l a r .body ( t ) i s X 31 ,000. v i s i b l e a t the. t o p . G l u t - 0s0. f i x a t i o n . 0 PLATE 11 F i g u r e 24. Two v e r y l a r g e t u b u l a r b o d i e s ( t ) i n a s s o c i a t i o n w i t h a d i f f u s e m i t o c h o n d r i o n (M)". Very l i t t l e o f the concent r i c 1 ame 1 1 ae (C) r e m a i n s . G l u t - OsO^ f i x a t i o n . X 4 3 , 0 0 0 . F i g u r e 25- A l a r g e c e n t r a l t u b u l a r body ( t ) and c o n c e n t r i c l a m e l l a e (C) complex. G l u t - OsO^ f i x a t i o n . X 31 ,000. F i g u r e 26. A v a c u o l e - l i k e body bound by c o n c e n t r i c l a m e l l a e (C) and c o n t a i n i n g t u b u l a r b o d i e s ( t ) appears t o be r e l e a s i n g i t s c o n t e n t s i n t o the space between the plasma membrane (PM) and the w a l l (W) (arr o w , Y ) . Running p a r a l l e l w i t h the l o n g a x i s o f the hypha a number of m i c r o t u b u l e s X 48,000. (MT)' a r e e v i d e n t . G l u t - 0s0, f i x a t i o n . PLATE 12 F i g u r e 27- A br a n c h (B) f o r m i n g a t a s e p t a t e r e g i o n o f a hypha. G r a n u l e s (G) 300 A i n d i a m e t e r i n a c l u s t e r may r e p r e s e n t g l y c o g e n d e p o s i t s . F i x a t i o n , a m i x t u r e o f G l u t - OsO^ i n c a c o d y l a t e b u f f e r . X 20,000, F i g u r e 28. A d e g e n e r a t e a r e a . L a r g e e l o n g a t e m i t o c h o n d r i a (M) packed c l o s e l y t o g e t h e r next t o a l a r g e c o n c e n t r i c l a m e l l a e s t r u c t u r e ( C ) . In the c e n t e r o f the c o n c e n t r i c l a m e l l a e s t r u c t u r e a few t u b u l a r b o d i e s ( t ) can be seen. G l u t - OsO^ f i x a t i o n . X 3^,000. PLATE 13 F i g u r e 29- A n o t h e r d e g e n e r a t e a r e a . M i t o c h o n d r i a (M) a s s o c i a t e d w i t h v a c u o l e s (V) . M i t o c h o n d r i a appear t o be u n d e r g o i n g i n t e r n a l r e a r r a n g e m e n t . KMnO^ f i x a t i o n . X 27,000. F i g u r e 30. A septum (S) as i t appears i n KMnO. f i x e d m a t e r i a l . X 40,000. PLATE 14 F i g u r e 31 • F i g u r e 32. F i g u r e 33-Septum o r c r o s s w a l l (S) formed a c r o s s the hypha. L = l i p i d . F i x e d w i t h a m i x t u r e o f G l u t d y l a t e b u f f e r . X 44,000. OsO^ in caco-Septum (S) s e p a r a t i n g f u n c t i o n a l c e l l (upper p o r t i o n ) from d e g e n e r a t i n g c e l l . G l u t - OsO^ f i x a t i o n . X 31,000. O b l i q u e v i e w o f a septum (S) w h i c h r e v e a l s the o r i e n t a t i o n o f the w a l l f i b e r s . F i x e d w i t h a m i x t u r e o f G l u t - 0s0, i n c a c o d y l a t e b u f f e r . X 48,000. PLATE 15 F i g u r e 34. Degenerate hypha f i l l e d w i t h t u b u l a r b o d i e s ( t ) . G l u t - OsO, f i x a t i o n . X 40,000. F i g u r e 35- The w a l l o f a d e g e n e r a t e hypha appears to have b u r s t s p i l l i n g o ut the c o n t e n t s o f the hypha i n t o a r e g i o n between o t h e r empty hyphae ( a r r o w s ) . G l u t - OsO^ f i x a t i o n . X 24,000. hi APPENDIX A (A) Comp1ete Med i urn (20 ml Vogel s o l u t i o n 1 | 1 l i t r e D i s t i l l e d w a t e r (50 mg T r y p t o p h a n ( 5 gm C a s e i n h y d r o l y s a t e 2 ( ^ 5 gm Y e a s t e x t r a c t ( D i f c o ) 20 gm S u c r o s e or 10 gm D e x t r o s e 10 ml V i t a m i n s o l u t i o n 15 gm Agar (Ba c t o ) - add b e f o r e h e a t i n g . F i r s t - add t r y p t o p h a n t o d i l u t e d V o g e l ' s s o l u t i o n -Second - add the next two i n g r e d i e n t s . T h i r d - add sugar and agar and v i t a m i n s o l u t i o n . Heat and pour p l a t e s . (B) M i n i m a l Medium M i n i m a l medium c o n t a i n s o n l y V o g e l ' s s o l u t i o n , d i s t i l l e d w a t e r , agar and d e x t r o s e or s u c r o s e . (C) V o g e l ' s S o l u t i o n Add c h e m i c a l s s u c c e s s i v e l y w i t h s t i r r i n g 123 gm Na^ c i t r a t e 2 H 2 0 250 gm K H 2 P 0 / j anhydrous 100 gm NH^NO^ anhydrous 10 gm M g S 0 v 7 H 2 0 5 gm C a C l 2 . 2 H 2 0 (add w i t h s t i r r i n g ) 5 c c t r a c e element s o l u t i o n 750 cc D i s t i 1 l e d H 20 2 c c C h l o r o f o r m To be d i l u t e d 5 0 - f o l d w i t h d i s t i l l e d H 20 b e f o r e use. S t o r e a t room t e m p e r a t u r e . V i tarnin S o l u t i o n 100 mg T h i a m i n 50 mg R i b o f l a v i n 50 mg P y r i d o x i n 200 mg C a l c i u m p a n t o t h e n a t e 50 mg p-amino B e n z o i c a c i d 200 mg N i c o t i n i c a c i d 200 mg C h o l i n e c h l o r i d e 400 mg i nos i t o l 50 mg F o l i c a c i d Di s t i1 l e d H 20 t o 1 1i t r e . S t o r e i n f r o z e n s t a t e . Use 10 ml o f v i t a m i n s o l u t i o n / 1 i t r e s t e r i 1 e med i urn. » APPENDIX B A f i ve day o l d (actua1 s i z e ) . m y c e l i a l c o l o n y p r i o r t o f i x a t i o n APPENDIX C T o l u i d i n e Blue s t a i n i n g p r o c e d u r e . S e c t i o n s a p p r o x i m a t e l y i u a r e p i c k e d up w i t h a s m a l l c opper l o o p ( 1-2 mm d i a m e t e r ) , and t r a n s f e r r e d t o a drop o f w a t e r on a c l e a n g l a s s s l i d e . The s l i d e i s heated g e n t l y o v e r a flame u n t i l the w a t e r e v a p o r a t e s . Then a drop of the s t a i n (1% T o l u i d i n e b l u e , \% Borax) i s p l a c e d on the s e c t i o n s and heated g e n t l y f o r 1 t o 5 seconds u n t i l the edge o f the drop t u r n s p u r p l e . Wash o f f the e x c e s s s t a i n w i t h w a t e r and d e h y d r a t e r a p i d l y i n 35% and 100% e t h a n o l . Mount u s i n g s t a n d a r d mounting medium o r immersion o i l - and s e a l w i t h n a i l p o l i s h . APPENDIX D P r o p i o n i c - Iron Alum - Haematoxy1in (Henderson and Lu, 1968) The s t a i n i s p r e p a r e d from equal volumes o f two s t o c k s o l u t i o n s : (A) 1% h a e m a t o x y l i n and (B) 0.5% i r o n alum, both i n 50% p r o p i o n i c a c i d , and i s ready f o r use 1 day a f t e r m i x i n g . A s m a l l amount from a m y c e l i a l c o l o n y of U. h o r d e i was t e a s e d o u t on a g l a s s c o v e r s l i p and a l l o w e d t o a i r d r y . Then th e m a t e r i a l was h y d r o l y z e d w i t h IN HCl f o r 60 minutes a t room t e m p e r a t u r e . A f t e r washing t h o r o u g h l y w i t h d i s t i l l e d H^ O, a few drops o f the s t a i n were a p p l i e d t o the m a t e r i a l on the c o v e r s l i p , and t h e m a t e r i a l squashed i n t h e s t a i n and mounted on a g l a s s s l i d e f o r v i e w i n g . 

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